Bead beating tube and method for extracting deoxyribonucleic acid and/or ribonucleic acid from microorganisms

ABSTRACT

The present disclosure provides improved methods for bead beating and a bead beating system useful therefor. The bead beating system comprises a sample tube, beads, and a dry blocking agent, and methods for using the bead beating system to extract nucleic acids from cells containing the nucleic acids.

This application is a division of U.S. application Ser. No. 15/432,157,filed Feb. 14, 2017, which is a continuation of PCT application no.PCT/EP2017/051902, filed Jan. 30, 2017, which claims the prioritybenefit of European patent application no. EP16153540.6, filed on Jan.30, 2016, the contents of each of which are incorporated by reference intheir entireties.

1. BACKGROUND

Genetic testing and diagnosis of infectious diseases play an importantrole in the field of clinical microbiology. To overcome the bias andlimitations inherent with culture-based methodologies, genetic testingis being increasingly used to detect microorganisms contained in asample. In order that such genetic testing can be performed, cells needto be lysed to allow extraction of the DNA and/or the RNA from themicroorganisms in a concentration as high as possible.

Zhongtang Yu et al., 2004, “Improved extraction of PCR-quality communityDNA from digesta and fecal samples,” BioTechniques, Vol. 36(5):808-812,describe a method for isolating DNA from digesta and fecal samples. Inthe method, 0.25 g of a sample fluid taken from a cow'sgastro-intestinal system, which is assumed to contain microorganisms,namely bacteria, is first of all mixed with 1 mL of a buffer containing500 mM sodium chloride, 50 mM Tris hydrochloride, pH 8.0, 50 mMethylenediaminetetraacetic acid, and 4% sodium dodecyl sulfate.Moreover, 0.4 g sterile zirconia beads are introduced in the samplefluid. The mixture thus obtained is subsequently oscillated by means ofa Mini-BeadBeater™ in a sample tube for three minutes such that thebeads destroy the cell walls of the microorganisms. In this process, theDNA contained in the cells is released. The buffer also serves toprotect the DNA from degradation by DNases which are contained in thesample fluid. After performing the bead beating, impurities are removedfrom the sample by precipitation with ammonium acetate. The nucleicacids are obtained by precipitation with isopropanol. In a furthermethod step the DNA is digested sequentially with RNase and Proteinase Kand subsequently purified by means of a column.

The method has the disadvantage that it is relatively complex. Since thesample fluid is diluted by the adding of the buffer, the DNAconcentration in the sample decreases. Therefore, the method allows onlya limited detection accuracy and sensitivity.

Thus, there is a need for improved devices and methods for lysing cellsand extracting nucleic acids from microorganisms contained in a samplefluid.

2. SUMMARY

The present disclosure relates to improved bead beating tubes and beadbeating systems and methods. Without being bound by theory, theinventors believe that bead beating permits nucleic acids to dissolveinto solution. The present disclosure is based, in part, on thediscovery that the use of bead beating in nucleic acid extractionmethods results in nucleic acid loss due to absorption on the beads andthat such loss can be prevented by the appropriate amount of blockingagent to block binding sites on the beads. The present disclosure isalso based on the recognition that although some lysis bufferspreviously used in bead beating may contain reagents that act asblocking agents, such reagents may be used in much lower quantity thanwas typically used. Further, the present disclosure is also based on therecognition that some biological samples, such as blood, inherentlycontain blocking agents and accordingly may undergo bead beating in theabsence of additives such as lysis buffers.

In certain aspects, the disclosure provides bead beating systems thatare suitable for use when bead beating samples that do not inherentlycontain blocking agents or contain blocking agents in amounts belowthose appropriate for effective blocking of nucleic acid absorption bybeads. The bead beating systems of the disclosure include dry blockingagents. Surprisingly, it has turned out that, when using beat beatingsystems of the disclosure for lysis of microorganisms and extraction ofdeoxyribonucleic acids (DNA) and/or ribonucleic acids (RNA) frommicroorganisms contained in a liquid sample, a larger extraction rate ofDNA and/or RNA can be achieved than when using a corresponding beadbeating tube which does not contain the blocking agent.

The bead beating systems of the disclosure can also circumvent the needto combine a sample with a lysis solution prior to bead beating,allowing the recovery of greater quantities of nucleic acids due to thelack of sample dilution.

In yet other aspects, the disclosure provides methods for performingbead beating to lyse cells and extract nucleic acids from biologicalsamples. The methods comprise performing bead beating on the biologicalsamples in the absence of lysis buffer. The bead beating methods canutilize the bead beating systems of the disclosure or standard beadbeating systems. When using standard bead beating systems, in someembodiments one or more blocking agents are added to the bead beatingsystems. In other embodiments, particularly where a biological samplenaturally includes one or more blocking agents, no blocking agent isadded prior to bead beating.

In certain embodiments, a bead beating system of the disclosure iscomposed a sample tube which comprises a container member with an innercavity, an aperture for filling a sample fluid containing microorganismsinto the inner cavity, and an attached or unattached closure for closingthe aperture, wherein a plurality of macroscopic particles are arrangedin the inner cavity which are adapted to mechanically destroy the cellwalls of the microorganisms contained in the sample fluid when thesample fluid is filled into the inner cavity and the bead beating tubeis subject to mechanical oscillations. Exemplary bead beading systemsare described in Section 4.1 and numbered embodiments 1 to 24 and 67 to75 below. Exemplary sample tubes that can be used in the bead beatingsystems are described in Section 4.1.1, exemplary blocking agents thatcan be used in the bead beating systems are described in Section 4.1.2,and exemplary beads that can be used in the bead beating systems aredescribed in Section 4.1.3.

The disclosure further relates to a method for lysing microoganisms toextract deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) fromthe microorganisms, wherein a sample fluid is provided which is assumedto contain the microorganisms, wherein a plurality of particles movablerelative to each other are introduced in the sample fluid and the samplefluid with the particles contained therein is oscillated such that theparticles are capable of mechanically destroying cell walls of themicroorganisms contained in the sample fluid. Exemplary samples fromwhich DNA can be extracted are described in Section 4.2 and exemplarysample pre-processing steps that can be used to prepare samples prior tonucleic acid extraction are described in Section 4.3. Exemplary methodsfor extracting nucleic acids from samples, for example from samplesdescribed in Section 4.2 or samples that have been pre-processed asdescribed in Section 4.3, are described in Section 4.4 and numberedembodiments 25 to 58 and 76 to 81 below. Kits useful for performing thenucleic acid extraction methods of the disclosure are described inSection 4.7 and numbered embodiments 59 to 66 below.

3. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: a side view of an exemplary bead beating system (1) comprising asample tube (2) and a closure cap (6).

FIG. 2: a plan view of the bead beating system shown in FIG. 1.

FIG. 3: a bottom view of the bead beating system shown in FIG. 1.

FIG. 4: a longitudinal section through the center plane of the beadbeating system shown in FIG. 1 with the closure cap removed from thesample tube, revealing an aperture (4) through which the inner cavity(3) of the sample tube is accessible. Beads (7) and a lyophilizedblocking agent (8) are shown in the inner cavity.

FIG. 5: a longitudinal section through the center plane of a beadbeating system shown in FIG. 1 filled with a sample fluid (5).

FIG. 6: shows intensity of PCR amplification product using DNA recoveredfrom C. albicans after bead beating as a template. Calbi144 and Calbi54are two different C. albicans target genes and HCO1-Rat87 is a negativecontrol gene. This study shows that urea has a blocking effect in beadbeating, facilitating the recovery of C. albicans DNA from PD fluid.

FIG. 7: shows intensity of PCR amplification product using DNA recoveredfrom C. albicans after bead beating as a template. Calbi144 and Calbi54are two different C. albicans target genes and HCO1-Rat87 is a negativecontrol gene. This study shows that RNA has a blocking effect in beadbeating, facilitating the recovery of C. albicans DNA from PD fluid.

FIG. 8: shows intensity of PCR amplification product using DNA recoveredfrom C. albicans after bead beating as a template. Calbi144 and Calbi54are two different C. albicans target genes and HCO1-Rat87 is a negativecontrol gene. An amount of C. albicans DNA corresponding to the maximumtheoretical yield of extraction was included as a template. This studyshows that C. albicans DNA can be recovered by bead beating blood in theabsence of additives.

FIG. 9: shows intensity of PCR amplification product using DNA recoveredfrom C. albicans after bead beating as a template. The genes probed fromare for detection of eubacteria (Eub), gram negative bacteria (Gng),gram positive bacteria (Gpo), E. coli (Eco), staphylocci (AllStaph),enterobacteriacae (Ent), S. aureus (Sau), mycobacteria (Myco). N03 Rat87 is a negative control, and CC is an internal control. An amount of S.aureus DNA corresponding to the maximum theoretical yield of extractionwas included as a template. This study shows that S. aureus DNA can berecovered by bead beating blood in the absence of additives.

FIG. 10: shows intensity of PCR amplification product using DNArecovered from C. albicans after bead beating as a template. The genesprobed from are for detection of eubacteria (Eub), gram negativebacteria (Gng), gram positive bacteria (Gpo), E. coli (Eco), staphylocci(AllStaph), enterobacteriacae (Ent), S. aureus (Sau), mycobacteria(Myco). N03 Rat 87 is a negative control, and CC is an internal control.An amount of E. coli DNA corresponding to the maximum theoretical yieldof extraction was included as a template. This study shows that E. coliDNA can be recovered by bead beating blood in the absence of additives.

4. DETAILED DESCRIPTION

Bead beating is a homogenization process used to lyse cells in order torelease their contents, including their nucleic acids (DNA and RNA).Samples are placed in tubes with the appropriate grinding beads andsubjected to high energy mixing. The samples are then typicallycentrifuged and the lysate recovered from above the beads. Typically,samples that are subject to bead beating are treated with lysis bufferto facilitate the release of DNA from cells.

The present disclosure relates to improved bead beating methods andsystems. The bead beating methods can be performed without the use oflysis buffer, simplifying the process of cell lysis and nucleic acidextraction and avoiding sample dilution while minimizing loss due toabsorption on beads.

The methods of the present disclosure relate to performing bead beatingusing the appropriate amount of blocking agents. The blocking agents canbe exogenous or endogenous to the sample. For example, blood containsurea, a reagent found to block binding of nucleic acids in a biologicalsample to bead beating beads. Accordingly, the present disclosureprovides methods of bead beating such samples without the addition ofexogenous blocking agents, although supplementation of endogenousblocking agents with exogenous blocking agents is also contemplated. Forbiological samples containing no or low amount of endogenous blockingagents, exogenous blocking agents can be used. The present disclosurefurther provides bead beating systems into which dry blocking agents areincorporated, which allows bead beating biological samples without theaddition of reagents that result in sample dilution.

Accordingly, the present disclosure provides bead beating systems thatinclude dry blocking agents. The present disclosure further providesmethods for performing bead beating to lyse cells in, and extractnucleic acids from, biological samples. The methods comprise performingbead beating on the biological samples in the absence of lysis buffer.The bead beating methods can utilize the bead beating systems of thedisclosure or standard bead beating systems. When using standard beadbeating systems, in some embodiments one or more blocking agents areadded to the bead beating systems. In other embodiments, particularlywhere a biological sample naturally includes one or more blockingagents, no blocking agent is added prior to bead beating.

Kits containing (or suitable for obtaining) the bead beating systems ofthe disclosure are also provided herein.

4.1 Bead Beating System

The present disclosure provides bead beating systems useful for celllysis and extraction of deoxyribonucleic acids (DNA) and ribonucleicacids (RNA) from cells (e.g., microorganisms) contained in a liquidsample. The bead beating systems of the disclosure comprise a sampletube, and beads and a dry blocking agent situated within the sampletube. Blocking agents that can be used in the bead beating systems ofthe disclosure include urea, guanidine salts, detergents, nucleotides,and oligonucleotides. Blocking agents are described in detail in Section4.1.2. Beads that can be used in the bead beating systems of thedisclosure include beads comprising a mineral and/or a metal. Beadssuitable for use in the bead beating systems of the disclosure aredescribed in Section 4.1.3.

4.1.1 Sample Tube

The bead beating systems of the disclosure comprise a sample tube havingan inner cavity that is accessible by an aperture and which canaccommodate beads, a blocking agent, and a liquid sample. The sampletube can be made of any biologically inert material (e.g., a plastic orborosilicate), and is preferably made of a plastic (e.g., polypropylene,polypropylene copolymer, or polycarbonate). As used herein, the term“tube” encompasses vials (e.g., grinding vials) and tubes (e.g., conicaltubes).

The bead beating system can include a closure for covering the apertureand sealing the sample tube. The closure can be fixed to the sample tube(e.g., a cap fixed to the sample tube by a hinge) or can be separablefrom the sample tube (i.e., a removable cap). The sample tube caninclude a threaded region that is adapted to engage a threaded cap. Thethreads can be on the inner or outer surface of the sample tube (e.g.,as shown in FIGS. 4-5).

Sample tubes that can be used in the bead beating system of thedisclosure are commercially available, for example, screw-cappolypropylene or microfuge tubes. Standard tube sizes can be used tomake the bead beating tubes and systems of the disclosure, for example0.5 mL, 1.7 mL, 2 mL, 4.5 mL, 7 mL, 15 mL, 50 mL or 250 mL.

4.1.2 Blocking Agent

The bead beating systems of the disclosure include dry blocking agents.As used herein, a “dry” or “dried” blocking agent is a blocking agentthat contains less than 20% moisture. In some embodiments, the dryblocking agent contains less than 15%, less than 10%, less than 5%, lessthan 4%, less than 3%, less than 2%, or less than 1% moisture.

The dry blocking agent is preferably long-term stable, i.e., the beadbeating system can be transported and stored without problems for alonger period without the blocking effect of the blocking agentdecreasing considerably. The dry blocking agent can be arranged looselyin the inner cavity of the container member, and/or part of or theentire inner cavity of the sample can be coated with a layer or a filmof the blocking agent. The blocking agent is preferably lyophilized. Theblocking agent can be lyophilized prior or after addition of the beadsto the tube.

Examples of blocking agents include one or more chaotropic agents (e.g.,urea, guanidine salts), one or more detergents, one or more nucleotides,one or more oligonucleotides, or any combination thereof. Details ofsuch exemplary blocking agents are set forth in Sections 4.1.2.1 to4.1.2.5, infra. The inclusion of blocking agents that contribute to thelysis of a microbial cell well (e.g., a detergent) may improve the yieldof nucleic acids prepared using the bead beating systems of thedisclosure and circumvent the need for combining a sample with a lysissolution prior to bead beating. Generally, the quantities of blockingagents used in the bead beating systems of the disclosure are lower thanis used in lysis solutions.

A bead beating system can be produced by lyophilizing a lysis solution(also referred to as an extraction buffer or lysis buffer) in a beadbeating tube. Lysis solutions containing blocking agents are known inthe art or can be purchased commercially.

In some embodiments, the blocking agent comprises one or more chaotropicagents. In some embodiments, the blocking agent comprises one or moredetergents. In some embodiments, the blocking agent comprises one ormore nucleotides. In some embodiments, the blocking agent comprises oneor more oligonucleotides. In some embodiments, the blocking agentcomprises one or more chaotropic agents and one or more detergents. Insome embodiments, the blocking agent comprises one or more chaotropicagents and one or more nucleotides. In some embodiments, the blockingagent comprises one or more chaotropic agents and one or moreoligonucleotides. In some embodiments, the blocking agent comprises oneor more detergents and one or more nucleotides. In some embodiments, theblocking agent comprises one or more detergents and one or moreoligonucleotides. In some embodiments, the blocking agent comprises oneor more nucleotides and one or more oligonucleotides. In someembodiments, the blocking agent comprises one or more chaotropic agents,one or more detergents, and one or more nucleotides. In someembodiments, the blocking agent comprises one or more chaotropic agents,one or more detergents, and one or more oligonucleotides. In someembodiments, the blocking agent comprises one or more chaotropic agents,one or more nucleotides and one or more oligonucleotides. In someembodiments, the blocking agent comprises one or more detergents, one ormore nucleotides, and one or more oligonucleotides. In some embodiments,the blocking agent comprises one or more chaotropic agents, one or moredetergents, one or more nucleotides, and one or more oligonucleotides.

The blocking agent can further comprise ethylenediaminetetraacetic acid(EDTA) and/or a sodium salt thereof. EDTA binds calcium, magnesium andiron and thus inactivates deoxyribonucleases and ribonucleases. Thismeasure also counteracts degradation of DNA and RNA contained in asample fluid processed using the bead beating system. Thus, higherdetection sensitivity and reproducibility of the measurement results isenabled during an examination of DNA and/or RNA extracted from cells(e.g., microorganisms) using a bead beating system of the disclosure.

The blocking agent can be arranged loosely in the inner cavity of thecontainer member, for example in powdered form. The powder can, forexample, be mixed with the beads in the sample, or can be arranged inthe sample tube as a layer above and/or below the beads. Alternativelyor additionally, the inner wall of the sample tube can be at leastpartially coated with a layer or a film of the blocking agent. The layeror film can be prepared by evaporating the liquid from an aqueousmixture comprising the blocking agent that has been added to the sampletube. In another embodiment, some or all of the beads in the beadbeating system can be coated with the blocking agent.

4.1.2.1 Chaotropic Agents

Chaotropic agents disrupt the structure of, and denature, macromoleculessuch as proteins and nucleic acids. Chaotropic solutes decrease the nethydrophobic effect of hydrophobic regions because of a disordering ofwater molecules adjacent to the protein. This solubilizes thehydrophobic region in the solution, thereby denaturing the protein. Thisis also directly applicable to the hydrophobic region in lipid bilayers;if a critical concentration of a chaotropic solute is reached (in thehydrophobic region of the bilayer) then membrane integrity will becompromised, and the cell will lyse.

Exemplary chaotropic agents that can be used as blocking agents areprovided below.

Urea: When the blocking agent comprises urea (or thiourea; as usedherein, unless the context dictates otherwise, the term urea includesthiourea), the amount of urea can be aligned with the amount of thesample fluid that is intended to be used with the bead beating systemsuch that the concentration of the urea dissolved in the sample fluidafter addition of the sample fluid to the sample tube ranges between 10and 100 grams per liter, between 50 and 100 grams per liter, between 20and 50 grams per liter, or between 25 and 35 grams per liter. Thus, withrespect to the foregoing embodiments, in case of a 2 mL tube to which 1mL of sample is to be added, the amount of urea used in the blockingreagent will be between 10 and 100 mg, between 50 and 100 mg, between 20and 50 mg, or between 25 and 35 mg, respectively, and in case of a 2 mLtube to which 0.8 mL of sample is to be added, the amount of urea usedin the blocking reagent will be between 8 and 80 mg, between 40 and 80mg, between 16 and 40 mg, or between 20 and 28 mg, respectively.

Salts: Certain salts can behave as chaotropic agents. Salts can havechaotropic properties by shielding charges and preventing thestabilization of salt bridges. Chaotropic salts include various salts ofguanidine, lithium and magnesium.

Guanidine Salts: Guanidine salts that can be used as blocking agents inthe bead beating systems of the disclosure include guanidine isocyanate,guanidine chloride, and combinations thereof.

Lithium Salts: Lithium salts that can be used as blocking agents in thebead beating systems of the disclosure include lithium perchlorate,lithium acetate, and combinations thereof.

Magnesium Salts: Magnesium salts that can be used as blocking agents inthe bead beating systems of the disclosure include magnesium chloride.

Detergents: Certain detergents can act as chaotropic agents, for examplesodium dodecyl sulfate (“SDS”). The use of detergents as blocking agentsis described in Section 4.1.2.3.

4.1.2.2 Creatinine

When the blocking agent comprises creatinine, the presence of thecreatinine in the inner cavity of the sample tube can counteractdegradation of DNA and/or RNA contained in a sample processed using thebead beating system. Furthermore, creatinine prevents the DNA and/or theRNA from nonspecifically binding to the beads and/or the inner wall ofthe sample tube. This enables larger yield when extracting DNA and/orRNA from cells (e.g., microorganisms) processed using the bead beatingtube system.

4.1.2.3 Detergents

Detergents that can be used as blocking agents in the bead beatingsystems of the disclosure include sodium dodecyl sulfate, sodiumlauroylsulfate sarcosinate, polyoxyethylene (20) sorbitan monolaurate,and combinations thereof. Polyoxyethylene (20) sorbitan monolaurate isalso known as polysorbate 20.

The amount of the detergent used in the blocking agent can be alignedwith the amount of the sample fluid that is intended to be used with thebead beating system. In certain embodiments, the concentration ofdetergent dissolved in the sample fluid after addition of the samplefluid to the sample tube ranges from 1 to 50 mg/mL, from 1 to 25 mg/mL,or from 25 to 50 mg/mL. Thus, with respect to the foregoing embodiments,in case of a 2 mL tube to which 1 mL of sample is to be added, theamount of detergent used in the blocking agent will range 1 to 50 mg,from 1 to 25 mg, or from 25 to 50 mg, respectively, and in case of a 2mL tube to which 0.8 mL of sample is to be added, the amount ofdetergent used in the blocking agent will range from 0.8 to 40 mg, from0.8 to 20 mg, or from 20 to 40 mg.

4.1.2.4 Nucleotides

Nucleotides that can be used in the bead beating systems of thedisclosure include ribonucleotides, deoxyribonucleotides, andcombinations thereof.

The nucleotides can be naturally occurring. Naturally occurringribonucleotides are adenosine monophosphate, guanosine monophosphate,cytosine monophosphate, and thymidine monophosphate. Naturally occurringdeoxyribonucleotides that can be used are blocking agents aredeoxyadenosine monophosphate, deoxyguanosine monophosphate,deoxycytosine monophosphate, and deoxythymidine monophosphate.

The nucleotides can be non-naturally occurring analogs of naturallyoccurring nucleotides. Examples of non-naturally occurring analogsinclude, but are not limited to, peptide nucleotides, locked nucleicacid (“LNA”) nucleotides (which contain bicyclic sugar moieties insteadof a deoxyribose or ribose sugars), those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), and those containing pendant moieties. Inspecific embodiments, the analog is 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxy-aminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine, or 7-deazaguanosine.

In some embodiments, the blocking agent comprises a mixture of at leasttwo, at least three, or four naturally occurring ribonucleotides. Inother embodiments, the blocking agent comprises a mixture of at leasttwo, at least three, or four naturally occurring deoxyribonucleotides.In yet other embodiments, the blocking agent comprises a mixture of atleast two, at least three, four or even more than four nucleotideanalogs. In yet other embodiments, the blocking agent comprises amixture of (a) naturally occurring ribonucleotides and naturallyoccurring deoxyribonucleotides, (b) naturally occurring ribonucleotidesand nucleotide analogs (c) naturally occurring deoxyribonucleotides andnucleotide analogs; or (d) naturally occurring ribonucleotides,naturally occurring deoxyribonucleotides and nucleotide analogs.

The amount of the ribonucleotides used in the blocking agent can bealigned with the amount of the sample fluid that is intended to be usedwith the bead beating system. In certain embodiments, the concentrationof ribonucleotides dissolved in the sample fluid after addition of thesample fluid to the sample tube ranges from 200 pg/ml to 20 μg/mL, from1 ng/ml to 15 μg/ml, or from 1 μg/ml to 10 μg/ml. Thus, with respect tothe foregoing embodiments, in case of a 2 mL tube to which 1 mL ofsample is to be added, the amount of ribonucleotides used in theblocking agent will range from 200 pg to 20 μg, from 1 ng to 15 μg, orfrom 1 μg to 10 μg, respectivley, and in the case of a 2 mL tube towhich 0.8 mL of sample is to be added, the amount of ribonucleotidesused in the blocking agent will range from 160 pg to 16 μg, from 0.8 ngto 12 μg, or from 0.8 μg to 8 μg.

4.1.2.5 Oligonucleotides

Oligonucleotides useful as blocking agents can be synthesized orgenerated from naturally occurring sources.

“Oligonucleotide” as referred herein does not connote the size of themolecule and means a polymeric form of nucleotides of any length.However, typically oligomers are no greater than 2,000 nucleotides,1,000 nucleotides, more typically are no greater than 500 nucleotides,and even more typically are no greater than 250 nucleotides. The termoligonucleotide includes double- and single-stranded DNA and RNA (but ispreferably at least partially single-stranded). It also includes knowntypes of modifications, for examples, labels which are known in the art,methylation, “caps”, substitution of one or more of the naturallyoccurring nucleotides with an analog (such as those described in Section4.1.2.4), such as, for example.

The oligonucleotides can be a mixture of different sizes and/or cancomprise ribonucleotides, deoxyribonucleotides, nucleotide analogs or acombination of two or more of the foregoing (e.g., a mixture ofribonucleotides and deoxyribonucleotides, a mixture of ribonucleotidesand nucleotides analogs, a mixture of deoxyribonucleotides andnucleotide analogs, or a mixture of ribonucleotides,deoxyribonuclotides, and nucleotide analogs). In some embodiments, theblocking agent comprises RNA.

Synthetic oligonucleotides that can be used in the bead beating systemsof the disclosure are typically 2 to 120 nucleotides long. In variousembodiments, the oligonucleotides are 2, 5, 8, 10, 15, 18, 25, 40, 50,80, 100 or 120 nucleotides long, or oligonucleotides having a lengthranging between any pair of the foregoing values, e.g., 2 to 100nucleotides long, 2 to 50 nucleotides long, 2 to 25 nucleotides long, 5to 40 nucleotides, 2 to 15 nucleotides long, 8 to 120 nucleotides long,8 to 80 nucleotides long, 10 to 100 nucleotides long, 15 to 50nucleotides long, 50 to 100 nucleotides long, 100 to 120 nucleotideslong, and so on and so forth.

Naturally occurring oligonucleotides can prepared by shearing DNA fromone or more naturally occurring sources, e.g., salmon sperm DNA, calfthymus DNA, herring sperm DNA, or combinations thereof.

The amount of the oligonucleotides used in the blocking agent can bealigned with the amount of the sample fluid that is intended to be usedwith the bead beating system. In certain embodiments, the concentrationof oligonucleotides dissolved in the sample fluid after addition of thesample fluid to the sample tube ranges from 200 pg/ml to 20 μg/mL, from1 ng/ml to 15 μg/ml, or from 1 μg/ml to 10 μg/ml. Thus, with respect tothe foregoing embodiments, in case of a 2 mL tube to which 1 mL ofsample is to be added, the amount of oligonucleotides used in theblocking agent will range from 200 pg to 20 μg, from 1 ng to 15 μg, orfrom 1 μg to 10 μg, respectivley, and in the case of a 2 mL tube towhich 0.8 mL of sample is to be added, the amount of ribonucleotidesused in the blocking agent will range from 160 pg to 16 μg, from 0.8 ngto 12 μg, or from 0.8 μg to 8 μg.

4.1.3 Beads

The bead beating system of the disclosure comprises beads that aremovable relative to each other when they are arranged in the samplefluid. As used herein, the term “bead” encompasses both spherical andnon-spherical particles. The beads can be mineral beads, for examplethose made of crystalline particles (e.g., zirconium, zircon (zirconiumsilicate) and zirconia (zirconium dioxide), quartz, aluminum oxide,silicon carbide (also known as carborundum), ceramic particles, glasses(e.g., silicon dioxide glass or silica), or combinations comprising oneor more of the foregoing (e.g., zirconia/silica beads orzirconia/yttrium beads). The beads can also be metal beads, for examplestainless steel beads or chrome-steel beads.

For recovery of bacterial DNA, the beads preferably include quartzparticles and/or zirconium particles. Such beads are commerciallyavailable in large amounts and are chemically inert with respect to DNAand RNA. Quartz and/or zirconium particles have a relatively highspecific weight, are hard and may have sharp edges. Therefore, they arewell suited for opening cell membranes when exposed to mechanicalvibrations.

The material and size of the beads for a given bead beating system canbe selected by those skilled in the art based upon the identity of thecell types in a fluid sample to be processed. Generally, the beads willrange from 50 μm to 3 mm in diameter. For extracting nucleic acids frombacterial cells, small to medium sized beads, typically made of glass orzirconium and ranging from 50 μm to 0.5 mm in diameter, can be used. Forextracting nucleic acids from larger microbial cells such as yeast,medium sized or large beads (e.g., beads with diameters of 0.5 mm, 1 mmor 1.5 mm beads, typically made of glass or zirconium) can be used.Beads of different sizes and compositions for processing different celltypes are commercially available. See, e.g., Benchmark Scientific,Product Note: Benchmark Bead Beating Guide, available atwww.denvillescientific.com/sites/default/files/Bead_Blasting_Notes.pdf.

The bead beating systems of the disclosure can include multiple types ofbeads and/or multiple sizes of beads of the same type, particularlywhere recovery of nucleic acid from multiple sources is desired (e.g.,bacterial and fungal DNA). Examples of bead beating systems withmultiple types of beads include systems with a combination of aluminumoxide beads and silicon carbide beads, a combination of ceramic beadsand silica beads, a combination of glass beads and zirconium oxidebeads, a combination of zirconia beads and aluminum oxide beads, or acombination of silicon carbide beads and glass beads. The differenttypes of beads can be the same or different sizes.

4.2 Samples

Examples of samples from which nucleic acids may be extracted using thebead beating methods of the disclosure include various fluid samples. Insome instances, the sample may be a bodily fluid sample from thesubject. The sample may include tissue collected from the subject. Thesample may include a bodily fluid, secretion, and/or tissue of asubject. The sample may be a biological sample. The biological samplemay be a bodily fluid, a secretion, and/or a tissue sample. Examples ofbiological samples may include but are not limited to, blood, serum,saliva, urine, gastric and digestive fluid, tears, stool, semen, vaginalfluid, interstitial fluids derived from tumorous tissue, ocular fluids,sweat, mucus, earwax, oil, glandular secretions, breath, spinal fluid,hair, fingernails, skin cells, plasma, nasal swab or nasopharyngealwash, spinal fluid, cerebrospinal fluid, tissue, throat swab, woundswab, biopsy, placental fluid, amniotic fluid, cord blood, emphaticfluids, cavity fluids, sputum, pus or other wound exudate, infectedtissue sampled by wound debridement or excision, cerebrospinal fluid,lavage, leucopoiesis specimens, peritoneal dialysis fluid, milk and/orother excretions.

The sample may be placed into a bead beating tube fresh from a subjector may have undergone some form of pre-processing (for example asdescribed in Section 4.3 below), storage, or transport prior toplacement in a bead beating tube. The sample may be added to a beadbeating system without undergoing intervention or much time.

A subject may provide a sample, and/or the sample may be collected froma subject. The subject can be a human or a non-human animal. The samplemay be collected from a living or dead subject. The animal can be amammal, such as a farm animal (e.g., cow, pig, sheep), a sport animal(e.g., horse), or a pet (e.g., dog or cat). The subject may be apatient, clinical subject, or pre-clinical subject. A subject may beundergoing diagnosis, treatment, and/or disease management or lifestyleor preventative care. The subject may or may not be under the care of ahealth care professional.

In some embodiments, the sample may be an environmental sample. Examplesof environmental samples may include air samples, water samples (e.g.,groundwater, surface water, or wastewater), soil samples, or plantsamples.

Additional samples may include food products, beverages, manufacturingmaterials, textiles, chemicals, therapies, or any other samples.

4.3 Sample Pre-Processing

In some instances, it might be advantageous to pre-process a sampleprior to processing in a bead beating system of the disclosure. Examplesof pre-processing steps that can be used prior to placing a sample in abead beating tube include filtration, distillation, extraction,concentration, centrifugation, inactivation of interfering components,addition of reagents, and the like, as discussed herein or otherwise asis known in the art.

It would be particularly advantageous to remove unwanted cell types andparticulate matter from biological samples prior to their placement intoa bead beating tube of the disclosure to maximize recovery of DNA from acell type of interest.

If the intent is to detect bacteria in a biological sample, then it isdesirable to pre-process the biological sample through a filter so thatparticulates and non-bacterial cells are retained on a filter whilebacterial cells (including their spores, if desired) pass through. A“filter,” as used herein, is a membrane or device that allowsdifferential passage of particles and molecules based on size. Typicallythis is accomplished by having pores in the filter of a particularnominal size. For instance, filters of particular interest for bacterialdetection applications have pores sufficiently large to allow passage ofbacteria but small enough to prevent passage of eukaryotic cells thatpresent in a sample of interest. Generally, bacterial cells range from0.2 to 2 μm (micrometers or microns) in diameter, most fungal cellsrange from 1 to 10 μm in diameter, platelets are approximately 3 μmdiameter and most nucleated mammalian cells are typically 10 to 200 μmin diameter. Therefore, filter pore sizes of less than 2 μm or less than1 μm are particularly suitable for removing non-bacterial cells from abiological sample if detection of bacteria is intended.

In addition to or in lieu of a filtration step, a biological sample canbe subject to centrifugation to remove cells and debris from a sampleprior to bead beating. Centrifugation parameters that precipitateeukaryotic but not bacterial cells are known in the art. The supernatantcan then be filtered if desired.

The filtrate generated by a filtration step can be the “sample” andplaced into a bead beating tube according to the disclosure, or subjectto further pre-processing steps (e.g., concentration or dilution).

4.4 Bead Beating

As an initial step in nucleic acid preparation, a sample is placed intoa bead beating tube for bead beating. The bead beating step can utilizea bead beating system of the disclosure or a standard bead beatingsystem. In some instances, one or more exogenous blocking agents can beadded when using a standard bead beating system but, in the case ofbiological samples containing endogenous blocking agents, the additionof exogenous blocking agents is not necessary. When one or more blockingagents are added, they can be added to a bead beating tube prior toaddition of the sample, after the addition of the sample, or both thesample and the blocking agent(s) can be added simultaneously (forexample, the sample and the blocking agent(s) can be mixed and thentransferred to a bead beating tube).

After placing the sample in the bead beating tube, the tube is subjectto agitation to mechanically lyse the cells. Lysis can be achieved by acommon laboratory vortexer or a homogenizer. While processing time in avortexer is 3-10 times longer than that in a specialty homogenizer,vortexing works for easily disrupted cells and is inexpensive.

Many homogenizers suitable for bead beating are commercially availableand can be used with the beat beating systems of the disclosure.Exemplary homogenizers are the BeadBug and BeadBlaster 24 (BenchmarkScientific), PowerLyzer 24 (MO Bio Laboratories Inc.), FastPrep-24,FastPrep-24 5G and SuperFastPrep-1 (MP Biomedicals), andMini-Beadbeater-16 (BioSpec Products).

The homogenization parameters can be chosen according to themanufacturer's recommendations. Generally, the duration of mechanicaldisruption (e.g., bead beating) can be less than 1 sec, 1-5 sec, 5-10sec, 10-25 sec, 25-60 sec, 1 min-2 min, 2 min-5 min, 5 min or longer.The number of repetitions of mechanical disruption (e.g., number of beadbeating sessions) can be 2, 3, 4, 5, 6, 7, 7-10, 10 or more repetitions.The speed of disruption (e.g., speed or setting for bead beating) may beless than 50, 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-1500,1500-2000, 2000 or more rotations (oscillations) per minute.

4.5 Nucleic Acid Purification

After cell lysis, the fluid sample is separated from the beads, forinstance by removing the liquid sample from the sample tube by means ofa pipette or the like, leaving behind the beads, which deposit on thebottom of the sample tube.

Impurities that might interfere with analysis of the extracted DNA canbe removed, for example by precipitation of the impurities by knownmethods such as with ammonium acetate or using a commercial kit.

The nucleic acid can be recovered and washed and optionally furtherpurified. Recovery and/or purification of nucleic acid can be carriedout using routine methods such as by precipitation with isopropanol orusing a commercial kit or an automated instrument.

4.6 Nucleic Acid Analysis

The analysis of a sample for the presence of nucleic acids of interest(such as bacterial or fungal DNA) can be performed using any nucleicacid analysis method including, but not limited to amplificationtechnologies, polymerase chain reaction (PCR), isothermal amplification,reverse transcription polymerase chain reaction (RT-PCR), quantitativereal time polymerase chain reaction (Q-PCR), digital PCR, gelelectrophoresis, capillary electrophoresis, mass spectrometry,fluorescence detection, ultraviolet spectrometry, hybridization assays,DNA or RNA sequencing, restriction analysis, reverse transcription,NASBA, allele specific polymerase chain reaction, polymerase cyclingassembly (PCA), asymmetric polymerase chain reaction, linear after theexponential polymerase chain reaction (LATE-PCR), helicase-dependentamplification (HDA), hot-start polymerase chain reaction,intersequence-specific polymerase chain reaction (ISSR), inversepolymerase chain reaction, ligation mediated polymerase chain reaction,methylation specific polymerase chain reaction (MSP), multiplexpolymerase chain reaction, nested polymerase chain reaction, solid phasepolymerase chain reaction, or any combination thereof. Such technologiesare well known to those skilled in the art. Knowing the sequence of atarget nucleic acid enables the scientist to construct primers and/orprobes that allow amplification and/or detection of the target. PCRamplicons can also be sequenced to confirm their identity.

By means of the bead beating system in accordance with the disclosure itis possible to extract DNA and/or RNA from the microorganisms containedin the sample fluid in a concentration sufficient to directly examinethe DNA and/or RNA microbiologically with per se known methods. This mayin particular occur by contacting the sample fluid with receptorsimmobilized on a surface, which bind specifically to the DNA and/or RNAand/or to DNA and/or RNA components contained therein. The binding ofthe DNA and/or RNA and/or of the DNA and/or RNA components to thereceptors may be detected in a per se known manner by means of markers,in particular optical markers such as fluorescent dyes, and bequantified if necessary. Alternatively, DNA and/or RNA from themicroorganisms contained in the sample fluid can be amplified prior toexamination, for example, by PCR.

4.7 Kits

The present disclosure further provides kits containing (or suitable forobtaining) the bead beating systems of the disclosure. The kits cancomprise a sample tube, such as a sample tube described in Section4.1.1, one or more or blocking agents, such as those described inSection 4.1.2, and/or one or more types of beads, such as thosedescribed in Section 4.1.3. Each of the one or more blocking agents canbe pre-lyophilized and can be included within the tube or separatelyfrom the tube. Likewise, the beads can be included within the tube orseparately from the tube.

In some embodiments, the kit comprises a sample tube and one or moreblocking agents. In a further embodiment, the kit further comprises oneor more types of beads.

The kits of the disclosure can include one or more components for samplepre-processing, e.g., saline, one or more buffer solutions, a filter asdescribed in Section 4.3, etc.

The kits can include one or more oligonucleotides for amplifying DNAand/or RNA from one or more pathogens of interest (e.g., one or more ofMycobacterium tuberculosis, Mycobacterium avium subsp paratuberculosis,Staphylococcus aureus, methicillin resistant Staphylococcus aureus(MRSA), Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcusagalactiae, Haemophilus influenzae, Haemophilus parainfuluezae,Moraxella catarrhalis, Klebsiella pneumoniae, Escherichia coli,Pseudomonas aeruginosa, Acinetobacter sp., Bordetella pertussis,Neisseria meningitidis, Bacillus anthracis, Nocardia sp., Actinomycessp., Mycoplasma pneumoniae, Chlamydia pneumonia, Legionella species,Pneumocystis jiroveci, influenza A virus, cytomegalovirus, rhinovirus,Enterococcus faecium, Acinetobacter baumannii, Corynebacteriumamycolatum, Enterobacter aerogenes, Enterococcus faecalis CI 4413,Serratia marcescens, Streptococcus equi, and Candida albicans).

The kits can include one or more probes for detecting DNA and/or RNAfrom one or more pathogens of interest (e.g., one or more ofMycobacterium tuberculosis, Mycobacterium avium subsp paratuberculosis,Staphylococcus aureus, methicillin resistant Staphylococcus aureus(MRSA), Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcusagalactiae, Haemophilus influenzae, Haemophilus parainfuluezae,Moraxella catarrhalis, Klebsiella pneumoniae, Escherichia coli,Pseudomonas aeruginosa, Acinetobacter sp., Bordetella pertussis,Neisseria meningitidis, Bacillus anthracis, Nocardia sp., Actinomycessp., Mycoplasma pneumoniae, Chlamydia pneumonia, Legionella species,Pneumocystis jiroveci, influenza A virus, cytomegalovirus, rhinovirus,Enterococcus faecium, Acinetobacter baumannii, Corynebacteriumamycolatum, Enterobacter aerogenes, Enterococcus faecalis CI 4413,Serratia marcescens, Streptococcus equi, and Candida albicans). In anembodiment, the one or more probes comprise oligonucleotides labeledwith a fluorescent dye. Different probes can be labeled with differentdyes to enable simultaneous detection of multiple pathogens of interest.

5. EXEMPLARY BEAD BEATING SYSTEM

An exemplary bead beating system is shown in the Figures. A bead beatingsystem designated with reference character (1) in FIGS. 1 to 3 comprisesa sample tube (2) having an inner cavity (3) and a closable aperture(4). The sample tube is preferably made of a plastic, but may also bemade of another biologically inert material.

A sample fluid (5), which is assumed to contain microorganisms, may befilled into the inner cavity (3) of the sample tube (2) and removedtherefrom through the aperture (4). For closing the aperture (4) thesample tube has a closure (6) adapted to be conveyed to an open and aclosed position and being designed as a closure cap comprising an innerthread which is adapted to be screwed with an outer thread provided onthe edge region of the outer wall of the sample tube (2) which boundsthe aperture (4).

In the inner cavity (3), beads (7) (e.g., 2 grams of silicon dioxideand/or zirconium beads) are further arranged whose largest dimension isapproximately 100 μm on average. The beads (7) are available in the formof a loose bulk in which they are movable relative to each other. Alyophilized blocking agent (8) is also arranged in the inner cavity (3).

The beads (7) serve to mechanically destroy the cell walls of themicroorganisms contained in the sample fluid (5) when the sample fluid(5) is filled into the inner cavity (3) and the bead beating tube issubject to mechanical oscillations, for instance, ultrasonicoscillations. They may be generated with an oscillation generator whichis not illustrated in detail in the drawing and may be transferred tothe bead beating system (1). Such oscillation generator is commerciallyavailable under the designation MPBio bead beater from MP Biomedicals.Due to the destruction of the cell walls the nucleic acids (e.g., DNA)contained in the microorganisms are released and dissolved in the samplefluid (5).

6. CASE STUDIES

6.1 Use of Bead-Beating to Recover Pathogen DNA from Sputum

Mycobacterium tuberculosis (“MTB”) is an obligate pathogenic bacterialspecies in the family Mycobacteriaceae and the causative agent of mostcases of tuberculosis. Primarily a pathogen of the mammalian respiratorysystem, it infects the lungs. Evidence suggests mycobacteria are able toenter into a state of non-replication or dormancy (spore formation)during stress like nutrient deficiency or hypoxia. The formation ofspores makes it challenging to lyse bacterial cells to extract bacterialDNA.

Further complicating recovering of mycobacterial DNA is thatmycobacterium strains are typically isolated from sputum of infectedpatients. Sputum is thick, viscous and difficult to process. Most sputumspecimens for analysis contain various amounts of organic debris and avariety of contaminating, normal, or transient bacterial flora. Chemicaldecontamination/processing is typically used to reduce the viscosity andkill the contaminants while allowing recovery of the mycobacteria.

Samples suspected of containing MTB present a potential risk to theuser. Accordingly, a sputum sample may be pre-treated by heating and/orinclusion of reagents suitable for inactivating microbes present in thesample to mitigate this risk. Inactivation of microbes, such as MTB, maybe carried out by heating (e.g., 90° C., 5 min.) to denature activeproteins, enzymatic digestion of cell wall structures, mechanicaldisruption to physically disrupt or inactivate the cells, chemicaltreatment or a combination thereof.

A sputum sample (typically collected in volumes between 1-10 mL, 5-10 mLor greater) may be initially liquefied to reduce its viscosity andheterogeneity for consistent sample processing. Sputum samples present aparticular challenge. MTB in sputum is one of the most challenging celland sample types to process due to the lipid-rich hydrophobic cell wallof acid fast bacilli and the viscous, heterogeneous nature of sputum.Standard extraction methods for sputum typically start with a process ofsedimentation, which often involves the treatment with a mucolytic agentsuch N-acetyl-L-cysteine (NALC), zephiran-trisodium phosphate (Z-TSP),or benzalkonium followed by centrifugation, decanting, and resuspension.

Accordingly, when processing highly viscous samples, such as sputum, thesample may be subjected to chemical treatment in order to reduce theviscosity so that subsequent processing steps) are not impeded. In oneembodiment, liquefaction of sputum samples by chemical treatment withmucolytic agents is carried out for 20 minutes at 60° C. Sputum has aviscosity range from about 100-6,000 cP (mPas) with a shear rate at 90s−1. The viscosity, measured in mPas, is determined by shear strengthdivided by shear rate. Preferably, the sample is liquefied to reduce theviscosity of sputum by at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, or at least 99%.

Following resuspension, the sample is added to a bead beating system (asdescribed in Section 4.1) of the disclosure for cell lysis, as describedin Section 4.4. Following lysis and nucleic acid extraction (see Section4.5), MTB DNA can be analyzed by PCR amplification of MTB sequences andMTB drug resistance can be analyzed by amplification of drug resistancegenes. Sputum samples can be analyzed for other bacterial and viralpathogens, including but not limited to, Staphylococcus aureus,methicillin resistant Staphylococcus aureus (MRSA), Streptococcuspyogenes, Streptococcus pneumoniae, Streptococcus agalactiae,Haemophilus influenzae, Haemophilus parainfuluezae, Moraxellacatarrhalis, Klebsiella pneumoniae, Escherichia coli, Pseudomonasaeruginosa, Acinetobacter sp., Bordetella pertussis, Neisseriameningitidis, Bacillus anthracis, Nocardia sp., Actinomyces sp.,Mycoplasma pneumoniae, Chlamydia pneumonia, Legionella species,Pneumocystis jiroveci, influenza A virus, cytomegalovirus, andrhinovirus.

6.2 Use of Bead-Beating to Recover DNA from Veterinary Pathogens

Mycobacterium avium subsp paratuberculosis (MAP) is a veterinarypathogen that is responsible for causing paratuberculosis or Johne'sdisease (JD), a chronic granulomatous gastroenteritis in domestic andwild ruminants. MAP is transmitted in herds both directly through semen,milk, colostrum and in-utero and indirectly by oral route throughcontaminated feed, fodder, pasture, waters etc., (fecal oral route). JDhas a devastating effect on the livestock productivity (early cullingand reduced milk production) and livestock industry incurs huge economiclosses. JD control efforts have been hampered by MAP's persistencewithin soil and water as well as shedding by subclinical and clinicalcattle.

As mentioned in the preceding section, the formation of spores makes itchallenging to lyse mycobacterial cells to extract bacterial DNA.Accordingly, the bead beating systems of the disclosure can be used toimprove MAP recovery. For example, the bead beating systems can be usedto recover DNA from MAP present in milk, soil, feces, semen, etc.

Milk samples can be directly processed using the bead beating systems ofthe disclosure or can be pre-processed, e.g., to increase theconcentration of MAP, if present, in the sample. An exemplarypre-treatment method comprises centrifuging a sample of milk to providea cream fraction, a whey fraction, and a pellet. The cream fraction andthe pellet can be pooled and added to a bead beating system of thedisclosure for cell lysis.

Fecal samples can be pre-processed, for example, by preparing fecalsuspensions that can then be added to a bead beating system of thedisclosure for cell lysis. An exemplary fecal suspension can be preparedby mixing a fecal sample with water, saline or a buffer (e.g., sodiumphosphate, phosphate buffered saline, Tris, etc.), allowing the mixtureto settle, separating and then centrifuging the supernatant and,finally, resuspending the pellet in a suitable liquid such as water,saline, or a buffer.

Soil samples can be suspended in a liquid (e.g., water, saline, or abuffer) and then added to a bead beating system of the disclosure forlysis. Semen samples can be similarly pre-processed by combining a semensample with an amount of water, saline, or buffer prior to bead beating.

6.3 Use of Bead-Beating to Recover Pathogen DNA from Wounds

For detection of wound infections, a wound swab can be incubated in asolution of saline, a cell culture medium, or a sample preparationsolution from a commercial kit. The swab is typically incubated for aperiod of approximately 5 minutes-1 hour and more preferably 0.25-1 hour(e.g., 0.25 hour, 0.5 hour or 0.75 hour). A suitable volume of solutionis 200 μL to 10 mL and in particular embodiments is 500 μL, 1 mL, 2 mL,5 mL, 8 mL or selected from a range bounded by any two of the foregoingembodiments, (e.g., 1 mL to 5 mL, 500 μl to 10 mL, 200 μL to 8 mL, andso on and so forth. The solution can be periodically shaken or agitatedto promote the release of pathogenic cells from the swab. At the end ofthe incubation period the swab can be squeezed and discarded.

The solution can then be placed in a bead beating system of thedisclosure. Optionally, it is filtered and/or concentrated prior to beadbeating.

Pathogens that commonly infect wounds include, but are not limited E.coli, P. aeruginosa, E. faecium, S. aureus (including MRSA), K.pneumoniae, A. baumannii, C. amycolatum, E. aerogenes, E. faecalis CI4413, S. marcescens, S. equi and C. albicans. Accordingly, wound swabsamples can be analyzed for any of the foregoing organisms, singly or incombination.

6.4 Use of Bead-Beating to Recover Pathogen DNA from Peritoneal DialysisFluid

Peritoneal dialysis is a treatment for patients with severe chronickidney disease. This type of dialysis uses the patient's peritoneum inthe abdomen as a membrane across which fluids and dissolved substancesare exchanged from the blood. Fluid is introduced through a permanenttube in the abdomen and then flushed out in order to clear excessivesalts, uric acid and other waste substances. By osmosis across theperitoneal membrane, solutes are exchanged between the blood and thedialysis fluid. After a suitable time, the dialysis fluid is removedfrom the peritoneum and discarded. In this way, the blood becomesequilibrated and terminal metabolic products, such as uric acid, areprevented from indefinitely accumulating in the blood.

The primary complication of peritoneal dialysis is infection due to thepresence of a permanent tube in the abdomen. At least one site ofinfection is in the peritoneal space, leading to abdominal pain, cloudydialysis effluent and findings of pathogens on gram stains or incultures from within the catheter. Further, the tissue tunnel orexternal surfaces of the catheter are common infection points from touchcontamination. Infection at the tissue tunnel is most commonly due tomigration from skin sites. Infections may lead to peritonitis and insome cases death.

Peritoneal infections can be detected in peritoneal dialysis fluidremoved from individuals undergoing peritoneal dialysis. Although S.aureus and P. aeruginosa are responsible for the majority of infections,other bacteria (diphtheroids, anaerobic organisms, nonfermentingbacteria, streptococci, nontuberculous mycobacteria, Legionella, yeasts,and fungi) can also be involved. Accordingly, peritoneal dialysis fluidsamples can be analyzed for any of the foregoing organisms, singly or incombination.

Peritoneal dialysis fluid can be pre-processed by filtration andoptionally concentration as described in Section 4.3.

6.5 Use of Bead-Beating to Recover DNA from Wastewater Contaminants

Example 1 of U.S. Pat. No. 9,290,796, directed to the detection ofproblematic foaming and bulking bacterial species in biologicalwastewater treatment process, describes extracting DNA wastewatercollected from wastewater treatment plants using a bead beatingprotocol. The bead-beating protocol can be adapted to incorporate abead-beating system of the disclosure.

6.6 Use of Bead-Beating to Recover DNA from Food Pathogens

Liquid food samples can be pre-processed by filtration and/orcentrifugation as described above in Section 4.3 prior to bead-beating.For example, drinks can preferably be pre-processed by filtration tocollect microorganisms that may be present. The microorganisms collectedby filtration can optionally be washed and subjected to centrifugationprior to bead-beating. For detection of pathogens from solid foods, suchas meat or fish, a sample of the food can be swabbed to collectmicroorganisms from the surface of the food. The microorganisms can thenbe washed from the swab and then subjected to bead-beating.

In some cases, it can be desirable to pre-culture a food sample for aperiod of time in order to facilitate growth of pathogens (e.g.,Salmonella) that may be present on or in the sample prior tobead-beating. For example, a solid food sample can be ground using alaboratory blender (e.g., a Stomacher® circulator, Seward Limited, UK)and then cultured to facilitate microorganism growth (e.g., for 8 to 12hours). A sample of the culture can then be subjected to bead-beating.

6.7 Use of Bead-Beating to Recover Pathogen DNA from Blood

The development of rapid molecular diagnostic tests for human infectionsis the most highly rated priority of the World Health Organization forhealth improvement of the world population (Daar et al., 2002, Nat.Genet. 32:229-232). Severe blood infections are an important cause ofmorbidity and death in hospitalized patients worldwide and one of themost important challenges in critical care. For example, recentestimates of sepsis incidence are of 240 cases per 100 000 in the UnitedStates. The human and economic burden of sepsis is considerable (Grossiet al., 2006, Surg. Infect. (Larchmt) 7:S87-S91). Despite advances ininfectious diseases and critical care management and numerous attemptsto develop new treatments, sepsis mortality rate remains unacceptablyhigh, ranging from 20% to 50%. Recognizing the signs of severe bloodinfections and/or severe sepsis and making an early and accuratediagnosis are the key to improving care and increasing the survivalrate. Indeed, rapid diagnostics could increase patient survival byreducing the time interval between blood sampling and antimicrobialtherapy application.

For molecular techniques of detecting pathogens (e.g., PCR and DNAsequence analysis), adequate isolation of pathogen DNA is critical toensure successful detection. Various methods use lysis buffers,sometimes accompanied by physical disruption methods such asbead-beating, to improve recovery of pathogen DNA from blood.

The present disclosure takes advantage of the presence of naturallyoccurring blocking agents in blood and provides simplified methods forisolation of pathogen DNA from blood. The blood can be subject to beadbeating without being diluted with any buffer or other additive, using abead beating system of the disclosure (containing blocking agents) or atraditional bead beating system (lacking blocking agents). After beadbeating, standard methods can be used to recover pathogen DNA, such asthose described in Section 4.5. Optionally, the recovered pathogen DNAis then analyzed, for example by the methods described in Section 4.6.

Sepsis causing pathogens are typically bacterial or fungal. Commonbacterial causes of sepsis are gram-negative bacilli (for example, E.coli, P. aeruginosa, E. corrodens, and H. influenzae). Other bacteriaalso causing sepsis are S. aureus, Streptococcus species, Enterococcusspecies and Neisseria. Candida species are some of the most frequentfungi that cause sepsis. Accordingly, blood samples can be analyzed forany of the foregoing organisms, singly or in combination.

7. EXAMPLES 7.1 Example 1: Bead Beating Systems Result in DNA Loss

7.1.1 Materials

The materials used in this study are:

Bead beating tube: part number: ARY0007 OPS Diagnostics, 4.5 mLcryovial, with 2.0 gm of 100 μm SiO₂ beads.

PD Fluid: Peritoneal dialysis fluid containing glucose and a balance ofNa⁺, Ca²⁺, Mg²⁺.

BE Buffer: Taigen Bioscience. Containing 1 μg of RNA/μl.

Urea: ACS grade

7.1.2 Methods

Two (2) mL of PD fluid spiked with EDTA at a level of 10 mM and C.albicans at 5000 CFU/mL was the standard matrix. Twelve (12) differentcombinations of urea and BE buffer were added to the sample, as shown inTable 1 below:

TABLE 1 Samples Sam- BE ple Buff- no. Sample er Urea 1 PD fluid + EDTA +5000 CFU/mL C.albicans 0 0 2 PD fluid + EDTA + 5000 CFU/mL C.albicans 30μl 0 3 PD fluid + EDTA + 5000 CFU/mL C.albicans 75 μl 0 4 PD fluid +EDTA + 5000 CFU/mL C.albicans 0 23.3 g/L 5 PD fluid + EDTA + 5000 CFU/mLC.albicans 30 μl 23.3 g/L 6 PD fluid + EDTA + 5000 CFU/mL C.albicans 02.3 g/L 7 PD fluid + EDTA + 5000 CFU/mL C.albicans 30 μl 2.3 g/L 8 PDfluid + EDTA + 5000 CFU/mL C.albicans 0 0.23 g/L 9 PD fluid + EDTA +5000 CFU/mL C.albicans 30 μl 0.23 g/L 10 PD fluid + EDTA + 5000 CFU/mLC.albicans 0 23.3 g/L 11 PD fluid + EDTA + 5000 CFU/mL C.albicans 30 μl23.3 g/L 12 PD fluid + EDTA + 5000 CFU/mL C.albicans 0 2.3 g/L

All samples were bead beaten for 45 seconds. Following bead beating, DNAwas isolated from the samples using the LabTurbo® 48 nucleic acidextraction system (Taigen Bioscience Corporation, Taipei, Taiwan) usingthe manufacturer's reagents and protocol. DNA isolated from 2.0 ml ofeach bead-beating sample was eluted in 100 μl of TE (Tris EDTA) buffer.The isolated DNA was subject to real time PCR amplification andquantification in order to determine the amount of target DNA in eachsample.

7.1.3 Results

The optical density ratio (which is an indicator of the purity of theisolated DNA) and corresponding DNA concentration for each sample isshown in Table 2 below:

TABLE 2 DNA Recovery DNA conc. Sample no. BE Buffer Urea A260/A280(ng/μL) 1 0 0 1.97 5.5 2 30 μl 0 3.02 54 3 75 μl 0 3.16 125 4 0 23.3 g/L2.45 18.7 5 30 μl 23.3 g/L 3.09 53 6 0 2.3 g/L 2.53 22 7 30 μl 2.3 g/L2.97 48.7 8 0 0.23 g/L 2.81 22.2 9 30 μl 0.23 g/L 3.05 51 10 0 23.3 g/L2.95 14.4 11 30 μl 23.3 g/L 3.01 56 12 0 2.3 g/L 2.93 22

The yield of DNA as reflected by intensity of signal obtained fromimaging of the signal from the array following PCR amplification andhybridization is graphically illustrated in FIG. 6 and FIG. 7.

7.1.4 Conclusion

In Sample 1, containing no blocking agent, the recovery of C. albicansDNA was only 5.5 ng/mL. The inclusion of urea as a blocking agentincreased recovery 3-4 fold and the inclusion of RNA as a blocking agentincreased recovery approximately 10-20 fold, and the yield approachedthe theoretical maximum. This increased recovery results in improvementsin C. albicans PCR gene amplification.

7.2 Example 2: Extraction of DNA Using a Bead Beating System of theDisclosure

7.2.1 Preparation of a Bead Beating System of the Disclosure

A stock blocking agent solution comprising 250 mg/mL urea, 25 mg/mLtetrasodium salt of ethylenediaminetetraacid (EDTA), 125 mg/mL adenosinemonophosphate, 125 mg/mL guanosine monophosphate, 125 mg/mL cytosinemonophosphate, 125 mg/mL thymidine monophosphate and 75 mg/mL sodiumlauroyl sarcosinate was prepared in TE (Tris/EDTA) buffer.

200 microliters of the stock solution was added to a 4.5- to 5-mL sampletube and taken to dryness by vacuum, resulting in the dried blockingagent being deposited in the lower region of the inner wall of thesample tube. The dried blocking agent is expected to be stable for atleast one year.

2.0 grams of 100 micrometer silicon dioxide beads were then added to thesample tube.

7.2.2 Use of a Bead Beating System of the Disclosure

6 mL of urine and 6 mL of peritoneal dialysis fluid were filteredthrough a 0.4 micrometer sterile filter and then 10,000 colony-formingunits per milliliter Staphylococcus aureus grown in a culture lab wereadded to the urine and peritoneal dialysis fluid filtrates.

2.9 mL of spiked urine and 2.9 mL of spiked peritoneal dialysis fluidwere transferred to sample tubes prepared as described in Section 7.2.1or to sample tubes containing 2.0 grams of 100 micrometer silicondioxide beads but lacking the blocking agent.

The tubes were capped and clamped in an MPBio bead beater and run for 30seconds at full power. The caps were removed and placed on the samplerack of a commercial DNA isolation device. 2 mL of each sample was takenout from each tube and DNA extraction agents were then added(LabTurbo®). DNA extraction was then performed. 100 microliters of DNAextract was obtained for each sample.

The amount of extracted S. aureus DNA in each sample was determined byreal time polymerase chain reaction according to Gillespie, et al.,2005, “Simultaneous Detection of Mastitis Pathogens, Staphylococcusaureus, Streptococcus uberis, and Streptococcus agalactiae by MultiplexReal-Time Polymerase Chain Reaction,” J. Dairy Sci. 88:3510-3518. 1microliter of each DNA extract was amplified in a 20 microliterreaction.

The samples processed using sample tubes lacking the blocking agent hada cycle threshold value of 32 for urine and of 38 for peritonealdialysis fluid, showing a low DNA recovery.

The samples processed using sample tubes with the blocking agent had acycle threshold value of 31 for urine and a cycle threshold value of 32for peritoneal dialysis fluid, showing an improved recovery for theperitoneal dialysis fluid under these conditions.

7.3 Example 3: Blood as a Blocking Agent

7.3.1 Overview

This study was performed in order to identify the optimal bead beatingconditions for blood samples. Culture-negative blood samples were spikedwith pathogens S. aureus, E. coli and C. albicans. A positive PCRcontrol was run. The concentration (genomic copies) of this control wasset as the theoretical 100% recovery of the introduced pathogen. Thisstudy demonstrated that is possible to extract DNA from blood pathogensusing bead beating in the absence of additives such as buffers,detergents, etc.

7.3.2 Materials

The materials specifically used in the study are:

Bead beating tube: part number: ARY0007 OPS Diagnostics, 4.5 mLcryovial, with 2.0 gm of 100 μm SiO₂ beads.

Blood: culture negative from healthy donors

S. aureus, E. coli and C. albicans cultures: all ATCC sourced

7.3.3 Methods

A series of thirty-eight (38) spiked blood samples were preparedconsisting of 3.0 mL of blood and 15 μL of saline cultures containingcultured pathogens. Nothing was added to each blood sample other thanthe saline culture containing the cultured pathogen. The beating tubeswere processed using a FastPrep®-24 homogenizer (MP Biomedicals) at aspeed of 6.5 m/s. DNA was then isolated from the samples using theLabTurbo® 48 nucleic acid extraction system (Taigen BioscienceCorporation) using the manufacturer's reagents and protocol. DNA wasamplified with 55 cycles of PCR and hybridized to an array which wasthen washed and imaged. The amounts of pathogens and bead beating timesare shown in Table 3 below:

TABLE 3 Samples Sample no. Sample Beads/Tubes Time 1 Blood/no spikeOPS/OPS 45 2 3 Blood + 5000 CFU/mL C. albicans OPS/OPS 45 4 5 Blood +5000 CFU/mL C. albicans OPS/OPS 50 6 7 Blood + 5000 CFU/mL C. albicansOPS/OPS 55 8 9 Blood + 5000 CFU/mL C. albicans OPS/OPS 60 10 11 Blood +5000 CFU/mL C. albicans OPS/OPS 65 12 13 Blood + 5000 CFU/mL S. aureusOPS/OPS 45 14 15 Blood + 5000 CFU/mL S. aureus OPS/OPS 50 16 17 Blood +5000 CFU/mL S. aureus OPS/OPS 55 18 19 Blood + 5000 CFU/mL S. aureusOPS/OPS 60 20 21 Blood + 5000 CFU/mL S. aureus OPS/OPS 65 22 23 Blood +100000 CFU/mL E. coli OPS/OPS 45 24 25 Blood + 100000 CFU/mL E. coliOPS/OPS 50 26 27 Blood + 100000 CFU/mL E. coli OPS/OPS 55 28 29 Blood +100000 CFU/mL E. coli OPS/OPS 60 30 31 Blood + 100000 CFU/mL E. coliOPS/OPS 65 32 33 Blood + 5000 CFU/mL C. albicans MPBio/MPBio 60 34 35Blood + 5000 CFU/mL S. aureus MPBio/MPBio 60 36 37 Blood + 100000 CFU/mLE. coli MPBio/MPBio 60 38

For the three different pathogens at the given spike level the genomicoutput per μl from the DNA isolation was calculated. This genomic copyvalue was used as the 100% efficiency target.

7.3.4 Results

Results are graphically illustrated in FIGS. 8 to 10, which show yieldof DNA as reflected by intensity of signal obtained from imaging of thesignal from the array following PCR amplification and hybridization.FIG. 8 presents the C. albicans times series along with the 100% target.FIG. 9 presents the S. aureus time series along with the 100% target.FIG. 10 presents the E. coli time series along with the 100% target.

7.3.5 Conclusion

For the three different pathogens, there was no evidence of signal lossdue to pathogen DNA binding to the beads. In all cases at least 100% oftheoretical was demonstrated. In 2 cases more the 100% was recovered,possibly due to the presence of DNA from dead bacteria in the spike.

8. SPECIFIC EMBODIMENTS

The present disclosure is exemplified by the specific embodiments below.

1. A bead beating system comprising (i) a sample tube having an innercavity that is accessible by an aperture, (ii) beads, and (iii) a dryblocking agent, wherein the beads and dry blocking agent are locatedwithin the inner cavity of the sample tube.

2. The bead beating system of embodiment 1, wherein the blocking agentcomprises one or more chaotropic agents, creatinine, one or morenucleotides, one or more oligonucleotides, or a combination thereof.

3. The bead beating system of embodiment 2, in which the blocking agentcomprises one or more chaotropic agents comprising urea, one or moreguanidine salts, one or more lithium salts, one or more magnesium salts,one or more detergents, or a combination thereof.

4. The bead beating system of embodiment 3, in which the blocking agentcomprises one or more guanidine salts comprising guanidine isocyanate,guanidine chloride, or a combination thereof.

5. The bead beating system of embodiment 3 or embodiment 4, in which theblocking agent comprises one or more lithium salts comprising lithiumperchlorate, lithium acetate, or a combination thereof.

6. The bead beating system of any one of embodiments 3 to 5, in whichthe blocking agent comprises magnesium chloride.

7. The beating system of any one of embodiments 3 to 6, in which theblocking agent comprises one or more detergents comprising sodiumdodecyl sulfate, sodium lauroylsulfate sarcosinate, polyoxyethylene (20)sorbitan monolaurate, or a combination thereof.

8. The bead beating system of any one of embodiments 2 to 7, in whichthe blocking agent comprises one or more nucleotides comprisingnaturally occurring nucleotides, non-naturally occurring nucleotides, ora combination thereof.

9. The bead beating system of embodiment 8, in which the one or morenucleotides comprise one or more naturally occurringdeoxyribonucleotides, one or more naturally occurring ribonucleotides,or a combination thereof.

10. The bead beating system of embodiment 9, in which the blocking agentcomprises one or more deoxyribonucleotides comprising deoxyadenosinemonophosphate, deoxyguanosine monophosphate, deoxycytosinemonophosphate, deoxythymidine monophosphate, or a combination thereof.

11. The bead beating system of embodiment 9 or embodiment 10, in whichthe blocking agent comprises one or more ribonucleotides comprisingadenosine monophosphate, guanosine monophosphate, cytosinemonophosphate, thymidine monophosphate, or a combination thereof.

12. The bead beating system of any one of embodiments 2 to 11, in whichthe blocking agent comprises one or more oligonucleotides comprisingribonucleotides, deoxyribonucleotides, nucleotide analogs, or acombination thereof.

13. The bead beating system of embodiment 12, in which the blockingagent comprises one or more oligonucleotides each of which isindependently 2 to 120 nucleotides long, 2 to 100 nucleotides long, 2 to50 nucleotides long, 2 to 25 nucleotides long, 5 to 40 nucleotides, 2 to15 nucleotides long, 8 to 120 nucleotides long, 8 to 80 nucleotideslong, 10 to 100 nucleotides long, 15 to 50 nucleotides long, 50 to 100nucleotides long, or 100 to 120 nucleotides long.

14. The bead beating system of embodiment 12 or embodiment 13, in whichthe oligonucleotides comprise DNA and/or RNA.

15. The bead beating system of any one of embodiments 1 to 14, in whichthe inner cavity of the sample tube is at least partially coated with alayer or film of the blocking agent.

16. The bead beating system of any one of embodiments 1 to 15, in whichsome or all of the beads are partially or fully coated with a layer orfilm of the blocking agent.

17. The bead beating system of any one of embodiments 1 to 16,comprising a powder containing the blocking agent.

18. The bead beating system of embodiment 17, wherein the powder is alyophilized powder containing the blocking agent.

19. The bead beating system of any one of embodiments 1 to 18, whereinthe beads are adapted to lyse bacteria, yeast, filamentous fungi,spores, plant cells, or animal cells.

20. The bead beating system of any one of embodiments 1 to 19, whereinthe beads comprise mineral beads, ceramic beads, glass beads, metalbeads, or a combination thereof.

21. The bead beating system of embodiment 20, wherein the beads comprisezirconium beads, zircon beads, zirconia beads, quartz beads, aluminumoxide beads, silicon carbide beads, ceramic beads, silicon dioxide glassbeads, stainless steel beads, chrome steel beads, or a combinationthereof.

22. The bead beating system of any one of embodiments 1 to 21, in whichthe beads have a diameter ranging from 50 μm to 3 mm.

23. The bead beating system of any one of embodiments 1 to 22, furthercomprising ethylenediaminetetraacetic acid (EDTA) and/or a sodium saltthereof located within the inner cavity of the sample tube.

24. The bead beating system of any one of embodiments 1 to 22, furthercomprising creatinine located within the inner cavity of the sampletube.

25. A method for lysing cells (e.g., to extract nucleic acids from thecells) contained in a liquid sample, comprising agitating the liquidsample within the sample tube of the bead beating system according toany one of embodiments 1 to 24 under conditions sufficient to lyse thecells.

26. A method for lysing cells (e.g., to extract nucleic acids from thecells) contained in a liquid sample containing an exogenous blockingagent, comprising agitating the liquid sample within a bead beatingsystem comprising a bead beating tube and beads in the absence of lysisbuffer and/or additives.

27. A method for lysing cells (e.g., to extract nucleic acids from thecells) contained in a liquid sample that has not been incubated withlysis buffer, comprising agitating the liquid sample and one or moreblocking agents within a bead beating system comprising a bead beatingtube and beads, optionally wherein the one or more blocking agents is ablocking agent present in a bead beating tube of any one of embodiments1 to 24.

28. The method of embodiment 26 or embodiment 27, wherein the beads areadapted to lyse bacteria, yeast, filamentous fungi, spores, plant cells,or animal cells.

29. The method of any one of embodiments 26 to 28, wherein the beadscomprise mineral beads, ceramic beads, glass beads, metal beads, or acombination thereof.

30. The method of embodiment 29, wherein the beads comprise zirconiumbeads, zircon beads, zirconia beads, quartz beads, aluminum oxide beads,silicon carbide beads, ceramic beads, silicon dioxide glass beads,stainless steel beads, chrome steel beads, or a combination thereof.

31. The method of any one of embodiments 26 to 30, in which the beadshave a diameter ranging from 50 μm to 3 mm.

32. The method of any one of embodiments 25 to 26, further comprising astep of placing the liquid sample within the sample tube prior toagitating the liquid sample.

33. The method of embodiment 25 or embodiment 32, wherein the agitatingcomprises subjecting the bead beating system to an oscillating motion.

34. The method of any one of embodiments 25 to 33, wherein the sample isa biological sample, an environmental sample, or a food product.

35. The method of embodiment 34, wherein the sample is a biologicalsample selected from blood, serum, saliva, urine, gastric fluid,digestive fluid, tears, stool, semen, vaginal fluid, interstitial fluid,fluid derived from tumorous tissue, ocular fluid, sweat, mucus, earwax,oil, glandular secretions, breath, spinal fluid, hair, fingernails, skincells, plasma, fluid obtained from a nasal swab, fluid obtained from anasopharyngeal wash, cerebrospinal fluid, a tissue sample, fluid ortissue obtained from a throat swab, fluid or tissue obtained from awound swab, biopsy tissue, placental fluid, amniotic fluid, peritonealdialysis fluid, cord blood, lymphatic fluids, cavity fluids, sputum,pus, microbiota, meconium, breast milk, or a sample processed, extractedor fractionated from any of the foregoing.

36. The method of embodiment 35, wherein the biological sample is urine,sputum or a sample processed, extracted or fractionated from urine.

37. The method of embodiment 35, wherein the biological sample is sputumor a sample processed, extracted or fractionated from sputum.

38. The method of embodiment 35, wherein the biological sample is awound swab or a sample processed, extracted or fractionated from a woundswab.

39. The method of embodiment 35, wherein the biological sample is bloodor a sample processed, extracted or fractionated from blood.

40. The method of embodiment 35, wherein the biological sample isperitoneal dialysis fluid or a sample processed, extracted orfractionated from peritoneal dialysis fluid.

41. The method of embodiment 34, wherein the sample is an environmentalsample selected from soil, groundwater, surface water, wastewater, or asample processed, extracted or fractionated from any of the foregoing.

42. The method of any one of embodiments 25 to 41, wherein the cellscomprise one or more pathogens.

43. The method of embodiment 42, wherein the one or more pathogenscomprise one or more bacterial pathogens, viral pathogens, fungalpathogens, or a combination thereof.

44. The method of embodiment 42 or embodiment 43, wherein the one ormore pathogens comprise one or more of Mycobacterium tuberculosis,Mycobacterium avium subsp paratuberculosis, Staphylococcus aureus,methicillin resistant Staphylococcus aureus (MRSA), Streptococcuspyogenes, Streptococcus pneumoniae, Streptococcus agalactiae,Haemophilus influenzae, Haemophilus parainfuluezae, Moraxellacatarrhalis, Klebsiella pneumoniae, Escherichia coli, Pseudomonasaeruginosa, Acinetobacter sp., Bordetella pertussis, Neisseriameningitidis, Bacillus anthracis, Nocardia sp., Actinomyces sp.,Mycoplasma pneumoniae, Chlamydia pneumonia, Legionella species,Pneumocystis jiroveci, influenza A virus, cytomegalovirus, rhinovirus,Enterococcus faecium, Acinetobacter baumannii, Corynebacteriumamycolatum, Enterobacter aerogenes, Enterococcus faecalis CI 4413,Serratia marcescens, Streptococcus equi, and Candida albicans.

45. The method of embodiment 42, in which the sample is sputum or asample processed, extracted or fractionated from sputum and the one ormore pathogens comprise one or more of Mycobacterium tuberculosis,Staphylococcus aureus, methicillin resistant Staphylococcus aureus(MRSA), Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcusagalactiae, Haemophilus influenzae, Haemophilus parainfuluezae,Moraxella catarrhalis, Klebsiella pneumoniae, Escherichia coli,Pseudomonas aeruginosa, Acinetobacter sp., Bordetella pertussis,Neisseria meningitidis, Bacillus anthracis, Nocardia sp., Actinomycessp., Mycoplasma pneumoniae, Chlamydia pneumonia, Legionella species,Pneumocystis jiroveci, influenza A virus, cytomegalovirus, andrhinovirus.

46. The method of embodiment 45, in which the one or more pathogenscomprise Mycobacterium tuberculosis.

47. The method of embodiment 42, in which the sample is milk or semen,or a sample processed, extracted or fractionated from milk, soil, feces,or semen, and the one or more pathogens comprises Mycobacterium aviumsubsp paratuberculosis.

48. The method of embodiment 42, in which the sample is a wound swab ora sample processed, extracted or fractionated from a wound swab and theone or more pathogens comprise one or more of E. coli, P. aeruginosa, E.faecium, S. aureus, K. pneumoniae, A. baumannii, C. amycolatum, E.aerogenes, E. faecalis CI 4413, S. marcescens, S. equi and C. albicans.

49. The method of embodiment 42, in which the sample is peritonealdialysis fluid or a sample processed, extracted or fractionated fromperitoneal dialysis fluid and the one or more pathogens comprise S.aureus and/or P. aeruginosa.

50. The method of any one of embodiments 25 to 49, wherein the liquidsample comprises cells from multiple species.

51. The method of any one of embodiments 25 to 50, further comprisingrecovering the liquid sample from the sample tube following cell lysis.

52. The method of embodiment 51, further comprising purifying thenucleic acids from the recovered liquid sample.

53. The method of any one of embodiments 25 to 52, further comprisinganalyzing one or more of the nucleic acids.

54. The method of embodiment 53, wherein the analysis is performed usinga microarray or by sequencing the one or more nucleic acids.

55. The method of embodiment 53 or embodiment 54, wherein a targetsequence is amplified prior to performing the analysis.

56. The method of embodiment 55, wherein the target sequence isamplified by PCR.

57. The method of any one of embodiments 25 to 56, wherein the nucleicacids comprise DNA.

58. The method of any one of embodiments 25 to 57, wherein the nucleicacids comprise RNA.

59. A kit for lysing cells (e.g., to extract nucleic acids from thecells) contained in a liquid sample, comprising the bead beating systemof any one of embodiments 1 to 24 and optionally: (i) one or morecomponents for preparing the liquid sample, (ii) one or moreoligonucleotides for amplifying one or more of the nucleic acids, (iii)one or more probes for detecting one or more of the nucleic acids, or(iv) any combination of (i) to (ii).

60. The kit of embodiment 59, which comprises one or more components forpreparing the liquid sample, and wherein the one or more components forpreparing the liquid sample comprise water, saline, a buffer, a filter,or a combination thereof.

61. The kit of embodiment 59 or embodiment 60, which comprises one ormore oligonucleotides for amplifying one or more of the nucleic acids.

62. The kit of any one of embodiments 59 to 61, which comprises one ormore probes for detecting one or more of the nucleic acids.

63. A kit for obtaining the bead beating system of any one ofembodiments 1 to 24, comprising a sample tube, beads, and a dry blockingreagent.

64. The kit of embodiment 63, in which the beads and/or dry blockingreagent are included in the kit separately from the sample tube.

65. The kit of embodiment 63, in which the beads and/or dry blockingreagent are included in the kit within the sample tube.

66. The kit of any one of embodiments 63 to 65, further comprising oneor more components for preparing a sample containing cells for nucleicacid extraction using the bead beating system, one or moreoligonucleotides for amplifying one or more nucleic acids, one or moreprobes for detecting one or more nucleic acids, or a combinationthereof.

67. A bead beating tube (1) comprising a sample tube comprising acontainer member (2) with an inner cavity (3), an aperture (4) forfilling a sample fluid (5) potentially containing microorganisms intothe inner cavity (3), and a closure (6) for closing the aperture (4),wherein a plurality of macroscopic, mineral particles (7) are arrangedin the inner cavity (3) which are adapted to mechanically destroy thecell walls of the microorganisms contained in the sample fluid (5) whenthe sample fluid (5) is filled into the inner cavity (3) and the beadbeating tube (1) is subject to mechanical oscillations, characterized inthat a blocking agent (8) comprising urea and/or at least one guanidinesalt and/or at least one detergent is arranged in dried form in theinner cavity (3) of the sample tube.

68. The bead beating tube (1) according to embodiment 67, characterizedin that the blocking agent (8) comprises deoxyadenosine monophosphate,deoxyguanosine monophosphate, deoxycytosine monophosphate,deoxythymidine monophosphate, or a mixture of at least two of thesemonophosphates.

69. The bead beating tube (1) according to embodiments 67 or 68,characterized in that the blocking agent (8) comprises adenosinemonophosphate, guanosine monophosphate, cytosine monophosphate,thymidine monophosphate, or a mixture of at least two of thesemonophosphates.

70. The bead beating tube (1) according to any of embodiments 67 to 69,characterized in that the at least one monophosphate contained in theblocking agent (8) is an oligonucleotide of at least 2 and at most 120nucleotides.

71. The bead beating tube (1) according to any of embodiments 67 to 70,characterized in that the at least one detergent comprises sodiumdodecyl sulfate, sodium lauroylsulfate sarcosinate and/orPolyoxyethylene (20) sorbitan monolaurate.

72. The bead beating tube (1) according to any of embodiments 67 to 71,characterized in that the dried blocking agent is arranged loosely inthe inner cavity (3) of the container member (2), preferably in powderedform.

73. The bead beating tube (1) according to any of embodiments 67 to 72,characterized in that the inner wall of the container member (2) is atleast partially coated with a layer or a film of the blocking agent.

74. The bead beating tube (1) according to any of embodiments 67 to 73,characterized in that ethylenediaminetetraacetic (EDTA) acid and/or asodium salt thereof is arranged in the inner cavity (3) of the sampletube.

75. The bead beating tube (1) according to any of embodiments 67 to 74,characterized in that creatinine is arranged in the inner cavity (3) ofthe sample tube.

76. Use of a bead beating tube (1) according to any of embodiments 67 to75 for extracting deoxyribonucleic acid and/or ribonucleic acid frommicroorganisms.

77. A method for extracting deoxyribonucleic acid and/or ribonucleicacid from microorganisms, wherein a sample fluid (5) is provided whichis assumed to contain the microorganisms, wherein a plurality of mineralparticles (7) movable relative to each other is introduced in the samplefluid (5) and the sample fluid (5) with the particles (7) containedtherein is oscillated such that the particles (7) are capable ofmechanically destroying cell walls of the microorganisms contained inthe sample fluid (5), characterized in that a blocking agent (8)comprising urea and/or at least one guanidine salt and/or at least onedetergent is introduced in the sample fluid (5) before and/or while theparticles (7) are oscillated.

78. The method according to embodiment 77, characterized in that theamount of urea is aligned with the amount of the sample fluid (5) suchthat the concentration of the urea dissolved in the sample fluid (5)ranges between 10 and 100 grams per liter, in particular between 20 and50 grams per liter, and preferably between 25 and 35 grams per liter.

79. The method according to embodiment 77 or 78, characterized in that,before and/or while the particles (7) are oscillated, at least one ofthe following monophosphates is introduced in the sample fluid (5):deoxyadenosine monophosphate, deoxyguanosine monophosphate,deoxycytosine monophosphate, deoxythymidine monophosphate.

80. The method according to any of embodiments 77 to 79, characterizedin that, before and/or while the particles (7) are oscillated, at leastone of the following monophosphates is introduced in the sample fluid(5): adenosine monophosphate, guanosine monophosphate, cytosinemonophosphate, thymidine monophosphate.

81. The method according to any of embodiments 77 to 80, characterizedin that creatinine and/or ethylenediaminetetraacetic acid (EDTA) and/ora sodium salt thereof are introduced in the sample fluid (5) beforeand/or while the particles (7) are oscillated.

9. CITATION OF REFERENCES

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.In the event that there is an inconsistency between the teachings of oneor more of the references incorporated herein and the presentdisclosure, the teachings of the present specification are intended.

9.1 Incorporation of EP16153540

The invention relates to a bead beating tube, comprising a sample tubecomprising a container member with an inner cavity, an aperture forfilling a sample fluid containing microorganisms into the inner cavity,and a closure for closing the aperture, wherein a plurality ofmacroscopic, mineral particles are arranged in the inner cavity whichare adapted to mechanically destroy the cell walls of the microorganismscontained in the sample fluid when the sample fluid is filled into theinner cavity and the beat beating tube is subject to mechanicaloscillations. The invention further relates to a method for extractingdeoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) frommicroorganisms, wherein a sample fluid is provided which is assumed tocontain the microorganisms, wherein a plurality of mineral particlesmovable relative to each other are introduced in the sample fluid andthe sample fluid with the particles contained therein is oscillated suchthat the particles are capable of mechanically destroying cell walls ofthe microorganisms contained in the sample fluid.

Genetic testing and diagnosis of infectious diseases are playing animportant role in the field of clinical microbiology. To overcome thebias and limitations inherent with cultivation-based methodologies,genetic testing is being increasingly used to detect microorganismscontained in a sample. In order that such genetic testing can beperformed, the DNA and/or the RNA first of all have/has to be extractedfrom the microorganisms in a concentration as high as possible. In amethod proposed by Zhongtang Yu et al. in “Improved extraction ofPCR-quality community DNA from digesta and fecal samples”,BioTechniques, Vol. 36, No. 5, p. 808-812 (May 2004), 0.25 g of a samplefluid taken from a cow's gastro-intestinal system are provided, which isassumed to contain microorganisms, namely bacteria. The sample fluid isfirst of all mixed with 1 mL of a buffer containing 500 mM sodiumchloride, 50 mM Tris hydrochloride, pH 8.0, 50 mMethylenediaminetetraacetic acid, and 4% sodium dodecyl sulfate.Moreover, 0.4 g sterile zircon beads are introduced in the sample fluid.The mixture thus obtained is subsequently oscillated by means of aMini-BeadBeater™ in a sample tube for three minutes such that the beadsdestroy the cell walls of the microorganisms. In this process, the DNAcontained in the cells is released. The buffer also serves to protectthe DNA from degradation by DNases which are contained in the samplefluid.

After performing the bead beating, impurities are removed from thesample by precipitation with ammonium acetate. The nucleic acids areobtained by precipitation with isopropanol. In a further method step theDNA is digested sequentially with RNase and Proteinase K andsubsequently purified by means of a column.

The method has the disadvantage that it is relatively complex. Since thesample fluid is diluted by the adding of the buffer, the DNAconcentration in the sample decreases. Therefore, the method allows onlya limited detection accuracy and sensitivity.

It is therefore an object to provide a bead beating tube and a method ofthe initially mentioned kind which enable deoxyribonucleic acids and/orribonucleic acids to be extracted quickly and in an amount sufficientfor detection from microorganisms contained in a sample fluid.

With respect to the beat beating tube this object is solved with thefeatures of embodiment 1′ described below. They provide that a blockingreagent which comprises urea and/or at least one guanidine salt and/orat least one detergent is arranged in dried form in the inner cavity ofthe sample tube.

Surprisingly, it has turned out that, when using such beat beating tubefor the extraction of deoxyribonucleic acids (DNA) and/or ribonucleicacids (RNA) from microorganisms contained in a sample fluid, a largerextracting rate of DNA and/or RNA can be achieved than when using acorresponding bead beating tube which does not contain the blockingreagent. It is assumed that the blocking reagent blocks binding sites ofthe mineral particles contained in the inner cavity of the sample tubeand thus prevents the binding of the DNA and/or RNA extracted from themicroorganisms to these binding sites. After the removing of theparticles from the sample fluid, which may, for instance be performed inthat the particles deposit on the bottom of the sample tube and thesample fluid located above the particles is removed from the sample tubeby means of a pipette or the like, a correspondingly high proportion ofDNA and/or RNA contained in the sample tube will then result in thesample fluid. The at least one detergent blocks binding sites at theinner wall of the sample tube to and prevents ribonucleic (RNA) acidand/or deoxyribonucleic acid (DNA) to bind to these binding sites. Thiswill also enable a high extraction rate of DNA and/or RNA.

Due to the presence of the blocking reagent it is even possible torenounce a dilution of the sample fluid with an extraction buffer beforebead beating. The sample fluid is in this case only diluted with theblocking reagent which may be present in the sample fluid inconsiderably lower concentration than is typically an extraction buffer.

Mineral particles are crystalline particles, ceramic particles, and/orglasses. The particles are movable relative to each other when they arearranged in the sample fluid. The particles preferably comprise quartzparticles and/or zircon particles. Such particles are cheaply availablein large amounts and are chemically inert with respect todeoxyribonucleic acid or ribonucleic acid. Quartz and/or particles havea relatively high specific weight, are hard and may have sharp edges.Therefore, they are well suited to open cell membranes when exposed tomechanical vibrations.

The dried blocking reagent is long-term stable, i.e. the bead beatingtube can be transported and stored without problems for a longer periodwithout the blocking effect of the blocking reagent decreasingconsiderably. The dried blocking reagent may be arranged loosely in theinner cavity of the container member, and/or the inner wall of thecontainer member may be coated with a layer or a film of the blockingreagent. The blocking reagent is preferably lyophilized.

By means of the bead beating tube in accordance with the invention it ispossible to extract DNA and/or RNA from the microorganisms contained inthe sample fluid in a concentration sufficient to directly examine theDNA and/or RNA microbiologically with per se known methods. This may inparticular occur by contacting the sample fluid with receptorsimmobilized on a surface, which bind specifically to the DNA and/or RNAand/or to DNA and/or RNA components contained therein. The binding ofthe DNA and/or RNA and/or of the DNA and/or RNA components to thereceptors may be detected in a per se known manner by means of markers,in particular optical markers such as fluorescent dyes, and bequantified if necessary.

The at least one guanidine salt comprises preferably guanidineisocyanate and/or guanidine chloride.

In a preferred embodiment of the invention the blocking reagentcomprises deoxyadenosine monophosphate, deoxyguanosine monophosphate,deoxycytosine monophosphate, deoxytymidine monophosphate, or a mixtureof at least two of these monophosphates. These monophosphates can blockbinding sites of the particles which deoxyribonucleic acid (DNA) canbind to.

In a preferred embodiment of the invention the blocking reagentcomprises adenosine monophosphate, guanosine monophosphate, cytosinemonophosphate, thymidine monophosphate, or a mixture of at least two ofthese monophosphates. These monophosphates can block binding sites ofthe particles which ribonucleic acid (RNA) can bind to. Ribonucleicacids are easier to degrade than deoxyribonucleic acid and are thereforebetter suited for a sample preparation.

It is of advantage if the at least one monophosphate contained in theblocking reagent is an oligonucleotide of at least 2 and at most 120 forexample RNA and/or DNA nucleotides. The at least one monophosphate isthen easy to produce synthetically.

In an expedient embodiment of the invention the at least one detergentcomprises sodium-dodecylsulafte, sodium lauroylsulfate sarcosinateand/or Polyoxyethylen-(20)-sorbitanmonolaurat.Polyoxyethylen-(20)-sorbitanmonolaurat is also known as polysorbat 20.This blocks binding sites at the inner wall of the sample tube to andprevents ribonucleic acid and/or deoxyribonucleic acid to bind to thesebinding sites.

In an advantageous embodiment of the invention the dried blockingreagent is arranged loosely in the inner cavity of the container member,preferably in powdered form. The powder can be mixed with the particles.

In a preferred embodiment of the invention the inner wall of thecontainer member is at least partially coated with a layer or a film ofthe blocking reagent. The blocking reagent is then captively connectedwith the container member.

It is of advantage if creatinine is arranged in the inner cavity of thesample tube. The presence of the creatinine in the inner cavity of thesample tube counteracts degradation of deoxyribonucleic acids and ofribonucleic acids contained in the sample fluid. Furthermore, thecreatinine prevents that the DNA and/or the RNA non-specifically bindsto the particles and/or the inner wall of the sample tube. This enableslarger yield when extracting DNA and/or RNA from microorganism containedin the sample fluid by means of the bead beating tube.

In an expedient embodiment of the invention ethylenediaminetetraaceticacid and/or a sodium salt thereof is arranged in the inner cavity of thesample tube. Ethylenediaminetetraacetic acid binds calcium, magnesiumand iron and thus inactivates deoxyribonucleases and ribonucleases. Thismeasure also counteracts degradation of deoxyribonucleic acids and ofribonucleic acids contained in the sample fluid. Thus, higher detectionsensitivity and reproducibility of the measurement results is enabledduring an examination of deoxyribonucleic acid and/or ribonucleic acidextracted from microorganisms by means of the bead beating tubes inaccordance with the invention.

With respect to the method the above-mentioned object is solved with thefeatures of embodiment 11′. They provide that a blocking reagent whichcomprises urea, at least one guanidine salt and/or at least onedetergent is introduced in the sample fluid before and/or while theparticles are oscillated.

In a preferred embodiment of the invention the amount of urea is alignedwith the amount of the sample fluid such that the concentration of theurea dissolved in the sample fluid ranges

-   -   between 10 and 100 grams per liter, in particular    -   between 20 and 50 grams per liter, and preferably    -   between 25 and 35 grams per liter.

This enables high yield when extracting the deoxyribonucleic acid and/orthe ribonucleic acid.

In a preferred embodiment of the invention the amount of detergent isaligned with the amount of the sample fluid such that the concentrationof the detergent dissolved in the sample fluid is between 1 and 50mg/ml.

In the following, embodiments of the invention will be explained indetail by means of the drawing.

A bead beating tube designated with 1 in FIGS. 1 to 3 comprises a sampletube comprising a container member 2 with an inner cavity 3 and aclosable aperture 4. The sample tube is preferably made of plastics, butmay also be made of another biologically inert material.

A sample fluid 5 which is assumed to contain microorganisms may befilled into the inner cavity 3 of the container member 2 and removedtherefrom through the aperture 4. For closing the aperture 4 the sampletube has a closure 6 adapted to be conveyed to an open and a closedposition and being designed as a closure cap comprising an inner threadwhich is adapted to be screwed with an outer thread provided on the edgeregion of the outer wall of the container member 2 which bounds theaperture 4.

In the inner cavity 3, 2 grams of silicon dioxide are further arrangedwhich comprise a plurality of particles 7 whose largest dimension isapproximately 100 μm on average. The particles 7 are available in theform of a loose bulk in which they are movable relative to each other.Instead of the particles 7 of silicon dioxide or in addition thereto,zirconium particles of appropriate size may also be contained in theinner cavity 3.

The particles 7 serve to mechanically destroy the cell walls of themicroorganisms contained in the sample fluid 5 when the sample fluid 5is filled into the inner cavity and the bead beating tube is subject tomechanical oscillations, for instance, ultrasonic oscillations. They maybe generated with an oscillation generator which is not illustrated indetail in the drawing and may be transferred to the bead beating tube 1.Such oscillation generator is commercially available under thedesignation MPBio bead beater with the Company MP Biomedicals. Due tothe destroying of the cell walls the deoxyribonucleic acid contained inthe microorganisms is released and dissolved in the sample fluid 5.

In the inner cavity 3 of the tube 1 a lyophilized blocking reagent 8 isalso arranged, which is prepared as follows:

5 milligram tetrasodium salt of ethylenediaminetetraacid (Na₄-EDTA), 25milligram adenosinmonophosphat, 25 milligram guanosinmonophosphat, 25milligram cyotsinmonophosphat, 25 milligram thymidinmonophosphat and 15mg sodium lauroyl sarcosinate are provided. In order to have theseregents stable they are dissolved in TE buffer in a 5× concentration.For reason of solubility the tetrasodium salt ofethylenediaminetetraacid is used as this dissolves well in water. 1milliliter then consists of 250 milligram Urea, 25 milligram tetrasodiumsalt of ethylenediaminetetraacid, 125 milligram adenosinmonophosphat,125 milligram guanosinmonophosphat, 125 milligram cyotsinmonophosphat,125 milligram thymidinmonophosphat and 75 mg sodium lauroyl sarcosinate.

200 microliter of this solution is put in the container member 2 andtaken to dryness by vacuum. The dried in blocking reagent 8 is stablefor at least one year. As can be seen in FIG. 4, the blocking reagent 8is applied on the inner wall of the container member 2 as a thin layerin a lower partial region of the inner cavity 3 which is spaced apartfrom the aperture 4.

EMBODIMENT

6 milliliter urine and peritoneal dialysis fluid are filtered through a0.4 micrometer sterile filter and then 10.000 colony-forming units permilliliter staphylococcus aureus grown in a culture lab are added.

Method of the Invention:

2.9 milliliter spiked urine and spiked peritoneal dialysis fluid aretransferred in the tube 1 described above. The tube is sealed by thescrew cap 6 and clamped in an MPBio bead beater and run for 30 secondsat full power. The closure 6 is removed and placed on the sample rack ofa commercial DNA isolation device. 2 milliliter of each sample are takenout and DNA extraction agents are then added (labturbo) and DNAextraction is performed. 100 microliter of DNA extract were produced.

Standard Method:

2.9 milliliter spiked urine and spiked peritoneal dialysis fluid aretransferred in a tube for bead beating with 2.0 gram of 100 micrometersilica (SiO₂) beads. The tube does not contain the blocking reagent 8and no further reagent are added. The tube is sealed by a closure andclamped in the MPBio bead beater and run for 30 seconds at full power.The closure is removed and placed on the sample rack of a commercial DNAisolation device. 2 milliliter of each sample are taken out and DNAextraction agents are then added (labturbo) and DNA extraction isperformed. 100 microliter of DNA extract were produced.

Detection of the Extracted DNA

The amount of extracted staphylococcus aureus DNA is determined by realtime polymerase chain reaction according to Gillespie, G. E. et al.:“Simultaneous Detection of Mastitis Pathogens, Staphylococcus aureus,Streptococcus uberis, and Streptococcus agalactiae by MultiplexReal-Time Polymerase Chain Reaction”, J. Dairy Sci. 88:3510-3518 (2005).1 microliter of extracted DNA was amplified in a 20 microliter reaction.The Standard method produced a cycle threshold value of 32 for urine andof 38 for peritoneal dialysis fluid showing a bad DNA recovery.

The method of the invention produced a cycle threshold value of 31 forurine and a cycle threshold value of 32 for peritoneal dialysis fluidshowing a much better recovery for the peritoneal dialysis fluid underthese conditions.

The disclosure is exemplified by the specific embodiments below.

-   1′. A bead beating tube (1) comprising a sample tube comprising a    container member (2) with an inner cavity (3), an aperture (4) for    filling a sample fluid (5) potentially containing microorganisms    into the inner cavity (3), and a closure (6) for closing the    aperture (4), wherein a plurality of macroscopic, mineral particles    (7) are arranged in the inner cavity (3) which are adapted to    mechanically destroy the cell walls of the microorganisms contained    in the sample fluid (5) when the sample fluid (5) is filled into the    inner cavity (3) and the bead beating tube (1) is subject to    mechanical oscillations, characterized in that a blocking reagent    (8) comprising    -   urea and/or    -   at least one guanidine salt and/or    -   at least one detergent    -   is arranged in dried form in the inner cavity (3) of the sample        tube.-   2′. The bead beating tube (1) according to embodiment 1′,    characterized in that the blocking reagent (8) comprises    -   deoxyadenosine monophosphate,    -   deoxyguanosine monophosphate,    -   deoxycytosine monophosphate,    -   deoxytymidine monophosphate,    -   or a mixture of at least two of these monophosphates.-   3′. The bead beating tube (1) according to embodiments 1′ or 2′,    characterized in that the blocking reagent (8) comprises    -   adenosine monophosphate,    -   guanosine monophosphate,    -   cytosine monophosphate,    -   thymidine monophosphate,    -   or a mixture of at least two of these monophosphates.-   4′. The bead beating tube (1) according to any of embodiments 1′ to    3′, characterized in that the at least one monophosphate contained    in the blocking reagent (8) is an oligonucleotide of at least 2 and    at most 120 nucleotides.-   5′. The bead beating tube (1) according to any of embodiments 1′ to    4′, characterized in that the at least one detergent comprises    sodium-dodecylsulafte, sodium lauroylsulfate sarcosinate and/or    Polyoxyethylen-(20)-sorbitanmonolaurat.-   6′. The bead beating tube (1) according to any of embodiments 1′ to    5′, characterized in that the dried blocking reagent is arranged    loosely in the inner cavity (3) of the container member (2),    preferably in powdered form.-   7′. The bead beating tube (1) according to any of embodiments 1′ to    6′, characterized in that the inner wall of the container member (2)    is at least partially coated with a layer or a film of the blocking    reagent.-   8′. The bead beating tube (1) according to any of embodiments 1′ to    7′, characterized in that ethylenediaminetetraacetic acid and/or a    sodium salt thereof is arranged in the inner cavity (3) of the    sample tube.-   9′. The bead beating tube (1) according to any of embodiments 1′ to    8′, characterized in that creatinine is arranged in the inner cavity    (3) of the sample tube.-   10′. Use of a bead beating tube (1) according to any of embodiments    1′ to 9′ for extracting deoxyribonucleic acid and/or ribonucleic    acid from microorganisms.-   11′. A method for extracting deoxyribonucleic acid and/or    ribonucleic acid from microorganisms, wherein a sample fluid (5) is    provided which is assumed to contain the microorganisms, wherein a    plurality of mineral particles (7) movable relative to each other is    introduced in the sample fluid (5) and the sample fluid (5) with the    particles (7) contained therein is oscillated such that the    particles (7) are capable of mechanically destroying cell walls of    the microorganisms contained in the sample fluid (5), characterized    in that a blocking reagent (8) comprising    -   urea and/or    -   at least one guanidine salt and/or    -   at least one detergent is introduced in the sample fluid (5)        before and/or while the particles (7) are oscillated.-   12′. The method according to embodiment 11′, characterized in that    the amount of urea is aligned with the amount of the sample fluid    (5) such that the concentration of the urea dissolved in the sample    fluid (5) ranges    -   between 10 and 100 grams per liter, in particular    -   between 20 and 50 grams per liter, and preferably    -   between 25 and 35 grams per liter.-   13′. The method according to embodiment 11′ or 12′, characterized in    that, before and/or while the particles (7) are oscillated, at least    one of the following monophosphates is introduced in the sample    fluid (5):    -   deoxyadenosine monophosphate,    -   deoxyguanosine monophosphate,    -   deoxycytosine monophosphate,    -   deoxytymidine monophosphate.-   14′. The method according to any of embodiments 11′ to 13′,    characterized in that, before and/or while the particles (7) are    oscillated, at least one of the following monophosphates is    introduced in the sample fluid (5):    -   adenosine monophosphate,    -   guanosine monophosphate,    -   cytosine monophosphate,    -   thymidine monophosphate.-   15′. The method according to any of embodiments 11′ to 14′,    characterized in that creatinine and/or ethylenediaminetetraacetic    acid and/or a sodium salt thereof are introduced in the sample fluid    (5) before and/or while the particles (7) are oscillated.

What is claimed is:
 1. A method for producing a lysate from cellscontained in a liquid sample, comprising agitating the liquid samplewithin a bead beating system in the absence of lysis buffer and in thepresence of one or more blocking agents under conditions sufficient tolyse the cells, said bead beating system comprising a bead beating tubeand beads.
 2. The method of claim 1, wherein the liquid sample containsan endogenous blocking agent.
 3. The method of claim 2, wherein theagitating is carried out in the absence additives.
 4. The method ofclaim 1, wherein the one or more blocking agents are exogenous to theliquid sample.
 5. The method of claim 2, wherein the beads: a) areadapted to lyse bacteria, yeast, filamentous fungi, spores, plant cells,or animal cells; b) comprise: i. mineral beads, ceramic beads, glassbeads, metal beads, or a combination thereof; ii. zirconium beads,zircon beads, zirconia beads, quartz beads, aluminum oxide beads,silicon carbide beads, ceramic beads, silicon dioxide glass beads,stainless steel beads, chrome steel beads, or a combination thereof; c)have a diameter ranging from 50 μm to 3 mm; or d) any combination ofa)-c).
 6. The method of claim 1, further comprising a step of placingthe liquid sample within the sample tube prior to agitating the liquidsample.
 7. The method of claim 1, wherein the agitating comprisessubjecting the bead beating system to an oscillating motion.
 8. Themethod of claim 1, wherein the sample is a biological sample, anenvironmental sample, or a food product.
 9. The method of claim 8,wherein the sample is a biological sample selected from blood, serum,saliva, urine, gastric fluid, digestive fluid, tears, stool, semen,vaginal fluid, interstitial fluid, fluid derived from tumorous tissue,ocular fluid, sweat, mucus, earwax, oil, glandular secretions, breath,spinal fluid, hair, fingernails, skin cells, plasma, fluid obtained froma nasal swab, fluid obtained from a nasopharyngeal wash, cerebrospinalfluid, a tissue sample, fluid or tissue obtained from a throat swab,fluid or tissue obtained from a wound swab, biopsy tissue, placentalfluid, amniotic fluid, peritoneal dialysis fluid, cord blood, lymphaticfluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk,or a sample processed, extracted or fractionated from any of theforegoing.
 10. The method of claim 9, wherein the biological sample is:a) urine, sputum or a sample processed, extracted or fractionated fromurine; b) sputum or a sample processed, extracted or fractionated fromsputum; c) a wound swab or a sample processed, extracted or fractionatedfrom a wound swab; d) blood or a sample processed, extracted orfractionated from blood; or e) peritoneal dialysis fluid or a sampleprocessed, extracted or fractionated from peritoneal dialysis fluid. 11.The method of claim 8, wherein the sample is an environmental sampleselected from soil, groundwater, surface water, wastewater, or a sampleprocessed, extracted or fractionated from any of the foregoing.
 12. Themethod of claim 1, wherein the cells comprise one or more pathogens. 13.The method of claim 12, wherein the one or more pathogens comprise: a)one or more bacterial pathogens, viral pathogens, fungal pathogens, or acombination thereof; or b) one or more of Mycobacterium tuberculosis,Mycobacterium avium subsp paratuberculosis, Staphylococcus aureus,methicillin resistant Staphylococcus aureus (MRSA), Streptococcuspyogenes, Streptococcus pneumoniae, Streptococcus agalactiae,Haemophilus influenzae, Haemophilus parainfuluezae, Moraxellacatarrhalis, Klebsiella pneumoniae, Escherichia coli, Pseudomonasaeruginosa, Acinetobacter sp., Bordetella pertussis, Neisseriameningitidis, Bacillus anthracis, Nocardia sp., Actinomyces sp.,Mycoplasma pneumoniae, Chlamydia pneumonia, Legionella species,Pneumocystis jiroveci, influenza A virus, cytomegalovirus, rhinovirus,Enterococcus faecium, Acinetobacter baumannii, Corynebacteriumamycolatum, Enterobacter aerogenes, Enterococcus faecalis CI 4413,Serratia marcescens, Streptococcus equi, and Candida albicans.
 14. Themethod of claim 12, in which the sample is sputum or a sample processed,extracted or fractionated from sputum and the one or more pathogenscomprise one or more of Mycobacterium tuberculosis, Staphylococcusaureus, methicillin resistant Staphylococcus aureus (MRSA),Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcusagalactiae, Haemophilus influenzae, Haemophilus parainfuluezae,Moraxella catarrhalis, Klebsiella pneumoniae, Escherichia coli,Pseudomonas aeruginosa, Acinetobacter sp., Bordetella pertussis,Neisseria meningitidis, Bacillus anthracis, Nocardia sp., Actinomycessp., Mycoplasma pneumoniae, Chlamydia pneumonia, Legionella species,Pneumocystis jiroveci, influenza A virus, cytomegalovirus, andrhinovirus.
 15. The method of claim 14, in which the one or morepathogens comprise Mycobacterium tuberculosis.
 16. The method of claim12, in which the sample is milk or semen, or a sample processed,extracted or fractionated from milk, soil, feces, or semen, and the oneor more pathogens comprises Mycobacterium avium subsp paratuberculosis.17. The method of claim 12, in which the sample is a wound swab or asample processed, extracted or fractionated from a wound swab and theone or more pathogens comprise one or more of E. coli, P. aeruginosa, E.faecium, S. aureus, K. pneumoniae, A. baumannii, C. amycolatum, E.aerogenes, E. faecalis CI 4413, S. marcescens, S. equi and C. albicans.18. The method of claim 12, in which the sample is peritoneal dialysisfluid or a sample processed, extracted or fractionated from peritonealdialysis fluid and the one or more pathogens comprise S. aureus and/orP. aeruginosa.
 19. The method of claim 1, further comprising recoveringthe lysate from the sample tube following cell lysis, and optionallyfurther comprising purifying the nucleic acids from the lysate.
 20. Themethod of claim 1, further comprising analyzing one or more of thenucleic acids, optionally wherein: a) the analysis is performed using amicroarray or by sequencing the one or more nucleic acids; b) a targetsequence is amplified prior to performing the analysis, optionally byPCR.
 21. A bead beating system comprising (i) a sample tube having aninner cavity that is accessible by an aperture, (ii) beads, and (iii) adry blocking agent, wherein the beads and dry blocking agent are locatedwithin the inner cavity of the sample tube.
 22. The bead beating systemof claim 21, wherein the blocking agent comprises: a) one or morechaotropic agents, creatinine, one or more nucleotides, one or moreoligonucleotides, or a combination thereof; b) one or more chaotropicagents comprising urea, one or more guanidine salts, one or more lithiumsalts, one or more magnesium salts, one or more detergents, or acombination thereof; c) one or more guanidine salts comprising guanidineisocyanate, guanidine chloride, or a combination thereof; d) one or morelithium salts comprising lithium perchlorate, lithium acetate, or acombination thereof; e) comprises magnesium chloride; f) one or moredetergents comprising sodium dodecyl sulfate, sodium lauroylsulfatesarcosinate, polyoxyethylene (20) sorbitan monolaurate, or a combinationthereof; g) one or more nucleotides comprising naturally occurringnucleotides, non-naturally occurring nucleotides, or a combinationthereof; h) one or more naturally occurring deoxyribonucleotides, one ormore naturally occurring ribonucleotides, or a combination thereof; i)one or more deoxyribonucleotides comprising deoxyadenosinemonophosphate, deoxyguanosine monophosphate, deoxycytosinemonophosphate, deoxythymidine monophosphate, or a combination thereof;j) one or more ribonucleotides comprising adenosine monophosphate,guanosine monophosphate, cytosine monophosphate, thymidinemonophosphate, or a combination thereof; k) one or more oligonucleotidescomprising ribonucleotides, deoxyribonucleotides, nucleotide analogs, ora combination thereof; or l) one or more oligonucleotides each of whichis independently 2 to 120 nucleotides long, 2 to 100 nucleotides long, 2to 50 nucleotides long, 2 to 25 nucleotides long, 5 to 40 nucleotides, 2to 15 nucleotides long, 8 to 120 nucleotides long, 8 to 80 nucleotideslong, 10 to 100 nucleotides long, 15 to 50 nucleotides long, 50 to 100nucleotides long, or 100 to 120 nucleotides long, optionally wherein theoligonucleotides comprise DNA and/or RNA.
 23. The bead beating system ofclaim 21, in which the inner cavity of the sample tube is at leastpartially coated with a layer or film of the blocking agent.
 24. Thebead beating system of claim 21, in which some or all of the beads arepartially or fully coated with a layer or film of the blocking agent.25. The bead beating system of claim 21, comprising a powder containingthe blocking agent, which is optionally a lyophilized powder containingthe blocking agent.
 26. The bead beating system of claim 21, wherein thebeads: a) are adapted to lyse bacteria, yeast, filamentous fungi,spores, plant cells, or animal cells; b) comprise: i. mineral beads,ceramic beads, glass beads, metal beads, or a combination thereof; orii. zirconium beads, zircon beads, zirconia beads, quartz beads,aluminum oxide beads, silicon carbide beads, ceramic beads, silicondioxide glass beads, stainless steel beads, chrome steel beads, or acombination thereof; c) have a diameter ranging from 50 μm to 3 mm; ord) any combination of a)-c).
 27. The bead beating system of claim 21,further comprising: a) ethylenediaminetetraacetic acid (EDTA) and/or asodium salt thereof located within the inner cavity of the sample tube;and/or b) creatinine located within the inner cavity of the sample tube.28. A method for lysing cells contained in a liquid sample, comprisingagitating the liquid sample within the sample tube of the bead beatingsystem according to claim 21 under conditions sufficient to lyse thecells.
 29. A kit for lysing cells contained in a liquid sample,comprising (i) the bead beating system of claim 21 and (ii) one or morecomponents for preparing the liquid sample, one or more oligonucleotidesfor amplifying one or more of the nucleic acids, one or more probes fordetecting one or more of the nucleic acids, or a combination thereof,optionally wherein the one or more components for preparing the liquidsample comprise water, saline, a buffer, a filter, or a combinationthereof.
 30. A kit for obtaining the bead beating system of claim 21,comprising a sample tube, beads, and a dry blocking reagent, optionally(i) in which the beads and/or dry blocking reagent are included in thekit separately from the sample tube, or (ii) in which the beads and/ordry blocking reagent are included in the kit within the sample tube.